The Journal of Immunology Lineage Divergence at the First TCR-Dependent Checkpoint: Preferential gd and Impaired ab T Cell Development in Nonobese Diabetic Mice
Ni Feng,Patricia Vegh,Ellen V.Rothenberg,and Mary A.Yui
The?rst TCR-dependent checkpoint in the thymus determines ab versus gd T lineage fate and sets the stage for later T cell differentiation decisions.We had previously shown that early T cells in NOD mice that are unable to rearrange a TCR exhibit a defect in checkpoint enforcement at this stage.To determine if T cell progenitors from wild-type NOD mice also exhibit cell-autonomous defects in development,we investigated their differentiation in the Notch-ligand–presenting OP9-DL1coculture system,as well as by analysis of T cell development in vivo.Cultured CD4and CD8double-negative cells from NOD mice exhibited major defects in the generation of CD4and CD8double-positive ab T cells,whereas gd T cell development from bipotent precursors was enhanced.Limiting dilution and single-cell experiments show that the divergent effects on ab and gd T cell development did not spring from biased lineage choice but from increased proliferation of gd T cells and impaired accumulation of ab T lineage double-positive cells.In vivo,NOD early T cell subsets in the thymus also show characteristics indicative of defective b-selection,and peripheral ab T cells are poorly established in mixed bone marrow chimeras,contrasting with strong gd T as well as B cell repopulation.Thus,NOD T cell precursors reveal divergent,lineage-speci?c differentiation abnormalities in vitro and in vivo from the?rst TCR-dependent developmental choice point,which may have consequences for subsequent lineage decisions and effector functions.The Journal of Immunology,2011,186:826–837.
T cells in all jawed vertebrates can be divided into two major subsets,those that use ab TCRs and those that use gd
TCR.These two major lineages are broadly similar,both requiring successful TCR gene rearrangement to develop and using many of the same effector genes.Yet accumulating evidence shows that their development is promoted by different balances of transcription factors,activating signals,and inputs from Notch–Delta interaction.Whereas most of these effects have been seen in the arti?cial context of targeted mutant mice,in this study we present evidence that there are unexpectedly sharp,divergent ab-normalities in ab T and gd T cell developmental programming in the unmanipulated genetic background of the autoimmune-prone NOD mouse.
All T cell lineages arise from the few multipotent precursor cells that migrate from bone marrow to the thymus,which provides the environmental signals,including Notch ligands and cytokines, needed for these progenitor cells to carry out the process of T lineage speci?cation and commitment.These cells undergo ex-tensive proliferation and an ordered series of differentiation stages characterized by a progressive induction of T cell identity genes and loss of stem cell and other genes permitting alternative fate choices(1).These stages are de?ned by speci?c changes in cell surface markers.In mice,the early double-negative(DN)stages lack CD4and CD8,as well as rearranged TCR,and differentiate ?rst from CD44+CD252c-Kit+DN1cells(or early thymic pre-cursors[ETPs])to CD44+CD25+c-Kit+DN2cells and then to CD442CD25+c-Kit lo DN3cells(2,3).TCR rearrangements are ongoing from the DN2through the DN3stage,and differentiation past the?rst T cell checkpoint at DN3requires successful rear-rangement and expression of a TCR b-chain or TCR g-and d-chains.If TCR b molecules are expressed,they pair with in-variant pre-T a molecules,triggering TCR signaling that results in b-selection(4–6).This is a process of rapid,major regulatory changes and extensive proliferation,coupled with effects on survival,culminating in differentiation to the CD4+CD8+double-positive(DP)stage.These DP cells undergo TCR a rearrange-ments and further selection and differentiation,based primarily but not solely on properties of the new ab TCR.T cells with strongly self-reactive TCRs are negatively selected and die, whereas cells with more moderate speci?city for self-antigens differentiate into a variety of effector populations including CD4 or CD8single-positive T cells,as well as unconventional cells like regulatory T cells,NKT cells,and CD8aa cells,before exiting to the periphery(reviewed in Ref.7).Alternatively,if g and d TCR chains are rearranged before a TCR b-chain,gd TCR expression triggers an alternative differentiation program,gd-selection,that typically results in a lower level of proliferation and no upregu-lation of CD4or CD8(reviewed in Ref.8).The nature of the receptor itself does not necessarily determine whether a cell will undergo ab T or gd T cell programming,but the intensity and timing of that signal is crucial,with pre-T a/TCR b typically giving weaker and gd TCR stronger signals(9,10).Thus,b-and gd-selection represent the?rst of several stages at which survival and further differentiation of a cell is dependent upon character-
Division of Biology,California Institute of Technology,Pasadena,CA91125 Received for publication August2,2010.Accepted for publication November9, 2010.
This work was supported by grants from the Juvenile Diabetes Research Foundation International,the National Institutes of Health(AI64590to M.A.Y.),the Al Sherman Foundation,the Louis A.Gar?nkle Memorial Laboratory Fund,the Vanguard Char-itable Endowment for Diabetes in Memory of Bently Pritsker,and the Albert Billings Ruddock Professorship(to E.V.R.).
Address correspondence and reprint requests to Mary A.Yui,Division of Biology, MC156-29,California Institute of Technology,1200E.California Boulevard,Pasa-dena,CA91125.E-mail address:yui@https://www.doczj.com/doc/794309662.html,
Abbreviations used in this article:B6,C57BL/6;BMC,bone marrow cell;DL1, Delta-like1;DL4,Delta-like4;DN,double-negative;DP,double-positive;ETP,early thymic precursors;FSC,forward scatter;HSA,heat shock Ag;ic,intracellular;ISP, immature single-positive;SP,single-positive.
Copyrightó2011by The American Association of Immunologists,Inc.0022-1767/11/$16.00 https://www.doczj.com/doc/794309662.html,/cgi/doi/10.4049/jimmunol.1002630 by guest on November 27, 2015 https://www.doczj.com/doc/794309662.html,/ Downloaded from
istics of the cell’s TCR and associated signaling apparatus.Many T cell identity genes,including those needed for TCR signaling,are turned on in the DN2–DN3stages for ?rst use at b -or gd -selection (1).For this reason,any fundamental genetically determined defect in T cells affecting signaling,survival,or proliferation could alter fate choices at this stage while also having an effect on,without being complicated by,subsequent lineage choices,positive and negative selection,and peripheral activation events.
Autoimmune type 1diabetes results from abnormal responses to self-antigens by peripheral T cells,due to both a failure of T cell self-tolerance mechanisms and breakdown of active T cell sup-pression of these autoreactive cells (reviewed in Ref.11).A number of studies have reported that different T lineages from autoimmune diabetes-prone NOD mice have defective numbers and/or responses to various stimuli relative to cells from other nonautoimmune mouse strains as possible factors contributing to disease.The T cell lineages implicated in type 1diabetes include CD4,CD8,NKT,and regulatory ab T cells,as well as gd T intraepithelial lymphocytes (11–16).However,by the time mature T cells initiate and propagate an autoimmune response to pe-ripheral tissues,they have already undergone a sequence of prior developmental choices and signaling encounters with APCs that may have contributed cumulatively to their abnormalities.There-fore,to shed light on the possible origins of inappropriate re-sponses of T cells in NOD mice,we have turned to the earliest stage when the TCR response machinery is ?rst used to dictate cell fate.This is during intrathymic development,at the checkpoint where commitment to the ab and gd T cell lineages is established.Although T cell populations in the thymi of NOD mice appear to be generally similar to those of other mouse strains in steady state (17,18),the early DN populations have not been investigated in great detail.Several studies have reported abnormalities in NOD thymic development.We previously reported that thy-mocytes from NOD.Prkdc scid /scid and NOD-Rag12/2mice appear to break through the b -selection checkpoint,resulting in the ab-errant development of DP cells (19).NOD thymi have also been shown to accumulate newly differentiated DP cells more slowly than conventional strains after dexamethasone-induced depletion of the DP compartment (20).Furthermore,a number of studies show defects in NOD DP cell responses to various stimuli,in-cluding responsiveness to Con A (21),apoptosis induced by glu-cocorticoids and gamma-irradiation (20)or anti-CD3ε(22),and responses to thymic selection signals (23–26).NOD mice also have defects in development of NKT cells from DP (17,27).All of these traits point to potential alterations in TCR signaling in NOD thymic T cells,which may have consequences for mature T cell activity (12).The nature of early T cell responses is set by the cellular signaling components,which in turn have effects on the thresholds for distinct responses including positive and nega-tive selection of ab TCR +DP cells (reviewed in Refs.7,28).The earliest stages of T cell development are dif?cult to assess in the presence of ongoing TCR rearrangements because they rep-resent only a small fraction of total thymocytes and because of the tremendous proliferation that occurs during differentiation from DN stages to DP,which can mask underlying developmental de-fects.In addition,steady-state proportions of thymic populations are affected by lineage decisions,cell death,and emigration (7).We have therefore used several approaches to compare the earliest stages of T cell development and lineage decisions in NOD to nonautoimmune C57BL/6(B6)mice.To investigate the intrinsic developmental potential of NOD T cell precursors,we took ad-vantage of the Notch-ligand–expressing OP9-DL1coculture sys-tem,which has been used extensively to elucidate the roles of Notch and other factors on the T cell developmental process in
mice and humans (29–32).We also carried out detailed ?ow cy-tometric analyses of early T cell developmental stages as well as tracking of population dynamics by BrdU labeling in vivo and in mixed bone marrow chimeras.Our ?ndings demonstrate that NOD early T cells exhibit impaired b -selection,whereas gd -selection is strongly enhanced.
Materials and Methods
Mice
NOD/ShiLtJ,C57BL/6J,BALB/cJ,NOD.Rag12/2,and B6.Rag12/2mice were obtained from The Jackson Laboratory (Bar Harbor,ME)and then bred and maintained in microisolator cages in our speci?c pathogen-free mouse colony in the Caltech Animal Resources facility (Pasadena,CA).The (NOD 3B6)F1.Rag12/2mice used for bone marrow chimeras were bred in our facility from the parent strains.Mice were used for these studies at 4–7wk of age.Euthanasia and animal care followed National Institutes of Health guidelines under protocols approved by the insti-tutional animal care and use committee.
Ab staining,cell sorting,and ?ow cytometric analysis
To isolate DN populations,mice were sacri?ced,their thymi were removed,and single-cell suspensions were made.Mature cells were depleted as previously described (33)by staining with biotinylated Abs to CD8a,TCR gd ,TCR b ,Gr1,Ter119,CD122,NK1.1,Dx5,and CD11c,after which the cells were incubated with streptavidin-coated magnetic beads and then passed through a magnetic column (Miltenyi Biotec,Auburn,CA).Eluted DN cells were either stained for direct ?ow cytometric analysis or for the sorting of speci?c DN populations into OP9cultures.ETP (CD252CD44hi c-Kit hi ),DN2(CD25+CD44hi c-Kit hi ),DN3a (CD25+CD44lo c-Kit lo CD27lo FSC lo ),and DN3b (CD25+CD44lo c-Kit lo CD27hi FSC hi )precursor cells,as well as gd T cells,were sorted from DN cells using a FACSAria with Diva software (Becton Dickinson Immunocy-tometry Systems,Mountain View,CA).For analysis of cocultures,cells were forcefully pipetted,?ltered through nylon mesh to remove stromal cell clumps,and stained for CD45to identify input cells plus various combinations of lineage identifying Abs.Cells were analyzed using a FACSCalibur (Becton Dickinson Immunocytometry Systems)or MACS-Quant (Miltenyi)and FlowJo software (Tree Star,Ashland,OR).Abs were purchased from eBioscience (San Diego,CA).For intracellular staining,DN thymocytes were stained with surface markers,?xed and perme-abilized using Cyto?x/Cytoperm Kit (BD),and stained with conjugated Abs to TCR b or TCR gd .
Cell culture
OP9-DL1cocultures were carried out as previously described (33,34).For T cell development,thymic DN cells were placed on monolayers of OP9-DL1,supplemented with 2.5ng/ml IL-7and 5ng/ml Flt3L.After 7d,cultures were transferred to new plates with fresh OP9-DL1cells and the medium replaced,supplemented with 1ng/ml IL-7.Human forms of the cytokines were used and obtained from Peprotech (Rocky Hill,NJ).Tissue culture media and FCS were obtained from Invitrogen/Life Technologies (Carlsbad,CA).For bone marrow cultures,cells were column-depleted of mature cells using biotinylated Abs to CD3e,CD4,CD8,CD19,Gr-1,Ter119,CD11b,NK1.1as described earlier.Cells (105)were plated on OP9-DL1monolayers in 10-cm tissue culture ?asks (Corning)with the addition of 10ng/ml IL-7and 5ng/ml Flt3L.After 6d,the bone marrow-derived cells,which are predominately DN1and DN2cells,were removed from the monolayers and replated on OP9-DL4cells with 2ng/ml IL-7.For proliferation assays,DN cell subsets were FACS sorted and then stained for 8min at 37?C with 5m M CFSE (Cell Trace CFSE Proliferation Kit;Invitrogen),in accordance with the manufacturer’s instructions,before they were placed in culture.
In vivo assays
For BrdU pulse-labeling of thymocytes,B6and NOD mice were injected i.p.with 1mg BrdU in 0.2ml PBS twice 4h apart.Mice were sacri?ced after 1,2,and 3d,and their thymocytes were stained with surface Abs followed by intracellular staining for BrdU–FITC in accordance with BrdU Flow Kit instructions (BD Biosciences).For mixed bone marrow chimeras,female 8-to 16-wk-old (NOD 3B6)F1.Rag12/2mice were irradiated with 400rad using a 137Cs source irradiator and retroorbitally injected with 53106bone marrow cells (BMCs)each from NOD and B6mice,mixed 1:1,and assayed between 5and 9wk after cell injection.To reduce the risk of infection,mice were housed using autoclaved cages,bedding,and food and
The Journal of Immunology
827
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
were treated with an antibiotic,Baytril (Bayer),in drinking water for several days before and 2wk after irradiation.
RNA extraction and real-time quantitative PCR analysis
RNA was isolated from sorted TCR gd +cells from B6and NOD mouse thymi and real-time quantitative PCR carried out as previously described (19).Brie?y,cells were lysed in Qiazol (Qiagen,Valencia,CA),RNA isolated using RNAeasy extraction kits (Qiagen),and cDNA reverse transcribed using random hexamers and SuperscriptIII (Invitrogen)fol-lowing the manufacturer’s instructions.cDNA samples were diluted and mixed with gene-speci?c primers and SYBR GreenER (Invitrogen)and run on an ABI Prism 7900HT Sequence Detector (ABI,Mountain View,CA).Primers used for this study were synthesized by Euro?ns MWG Operon (Huntsville,AL)and have been published previously (19,35,36)except for those for Sox13:forward,59-GAGAAGCTGCTGTCCAGT-GAC-39;reverse,59-GACCAAAAGCTGGAGTTCCTT-39.V g 1.1and J g 4primer sequences were obtained from Verykokakis et al.(37).Results were calculated using the D Ct method,normalizing all samples to Actb ex-pression.
Results
NOD DN cells are impaired in the generation and maintenance of ab T lineage DP cells,but not gd T cells,in OP9-DL1cocultures
Because steady-state thymic T cell populations re?ect a combi-nation of intrinsic and extrinsic factors,proliferation,cell death,and emigration,which can mask developmental differences,we wished to assess the intrinsic developmental potential of T cell precursors from B6,NOD,and BALB/c mice under the same environmental conditions using the OP9-DL1coculture system (30,34).Immature DN thymocytes were puri?ed by depletion of mature cells and placed in OP9-DL1coculture with IL-7and Flt3L.When analyzed on day 12of culture,B6and BALB/c DN cells were found to generate and maintain high percentages of DP cells as expected,whereas NOD DN cells produced a much lower proportion of DP cells under the same conditions (Fig.1A ,top panels ).In the same cultures,NOD DN cells produced a higher proportion of gd T cells relative to B6and BALB/c DN cells (Fig.1A ,bottom panels ).
To assess the kinetics of stage-speci?c ab versus gd lineage development of NOD and B6DN cells,we tracked the devel-opment of puri?ed intrathymic DN precursor subsets,including DN1/ETP (c-Kit hi CD252CD44hi ),DN2(c-Kit hi CD25+CD44hi ),and DN3a (c-Kit lo CD25+CD44lo CD27lo FSC lo )populations that retain both gd and ab T cell potential,as well as DN3b cells (c-Kit lo CD25+CD44lo CD27hi FSC hi ),which,as we previously showed for B6mice,have already completed TCR b rearrangement and pre-TCR signaling (34).Although NOD thymocytes also upreg-ulate CD27at b -selection,they do so to a lesser extent than B6cells (see later),so NOD and B6DN3b cells were enriched by sorting based on combined expression of relatively high levels of CD27and large size (high forward scatter [FSC],whereas DN3a cells were sorted based upon lower CD27and lower FSC (38).When precursor DN subsets from NOD and B6thymi were placed in OP9-DL1cocultures,neither B6nor NOD DN1/ETP and DN2cells generated DP cells by day 7(Fig.1B ).However,by day 14,B6DN1/ETP and DN2cell cultures contained abundant cells that had differentiated to the DP stage,whereas NOD DN1and DN2cells produced few if any DP cells.This is not due simply to a delay in differentiation,as NOD DP cell production did not increase with time (data not shown).
In contrast,DN3cultures from both B6and NOD thymi began to express CD4and/or CD8by day 7of OP9-DL1coculture (Fig.1B ),a time point when few DN1or DN2cells from either strain had turned on CD4or CD8.Con?rming the ab T cell lineage choice of the accumulated DP cells,all cultures that contained abundant
DP cells,including B6and NOD DN3a and DN3b at day 7,as well as all B6DN subsets at day 14,clearly expressed surface TCR b ,whereas those NOD and B6cultures with few DP cells express very little TCR b (Fig.1C ).At day 7,the percentage of DP from NOD DN3a cells was moderately lower in comparison with that of B6DN3a cells,and NOD DN3b cells appeared to produce fewer cells overall and a lower proportion of DP cells.By day 14,the differences were even more pronounced,with a very low proportion of DP cells in NOD DN3a cultures and very few sur-viving cells in the DN3b cultures.This latter result was con?rmed in further experiments when the ability of individual post–b -se-lection DN3b cells to generate DP cells was tested.Single DN3b cells were sorted from NOD and B6thymi and placed in OP9-DL1coculture for 7d.Although all individual NOD and B6DN3b cells proliferated and differentiated,fewer DP cells were generated in the NOD cultures (35.268.9,mean 6SEM)compared with that in B6cultures (179.5653.5)(p ,0.005,Mann–Whitney U test).These results suggest a possible loss of differentiation potential,proliferation,and/or viability in the generation of DP cells from DN cells sorted both before and after b -selection in the NOD thymus.
To identify the stages at which the initial differentiation of the NOD DN cells might be blocked,the same cultures were stained with CD44and CD25at day 7(Fig.1D ).Most of the cells from cultures seeded with DN3a and DN3b cells from either B6or NOD mice had progressed to DN4,or later stages,as shown by the downregulation of CD25and CD44by day 7,in agreement with their predominately DP phenotype.Although both B6and the NOD DN1and DN2cells differentiated successfully to the DN3stage,the NOD DN1and DN2cell cultures produced a much lower proportion of DN4and later cells (Fig.1D ),suggestive of a partial block at the b -selection checkpoint from NOD DN1and DN2cells.
In marked contrast to their poor production of DP cells,the DN1,DN2,and DN3a cultures from NOD thymocytes generated gd T cells very successfully (Fig.1E ).All of the NOD populations produced a higher proportion of gd T progeny than that of the corresponding B6precursors,with DN2cells producing the greatest differences:2.8%from B6and 21%from NOD DN2cultures on day 14.As expected,very few gd T cells were gen-erated from sorted DN3b cells from either strain,in agreement with our previously published results for B6mice (34).Thus,even though NOD DN1and DN2cells are extremely poor at passing through the b -selection checkpoint to generate DP ab T lineage cells in vitro,they are normal or enhanced in their ability to produce gd T lineage cells.
These results show that NOD precursors exhibit a lineage-speci?c intrinsic defect in development when placed in OP9-DL1cultures.DN3cells from NOD mice,even those that had undergone b -selection in the thymus,generate fewer DP cells than do B6DN3cells,and the earliest DN1and DN2T cell precursors sorted from NOD thymi are profoundly defective in their ability to go through b -selection in vitro.However,these cells retain the ability to produce abundant TCR gd +cells.
Prior thymic contact is not required for skewing of ab versus gd T cell fate from NOD cells
Although the highly skewed production of ab versus gd lineage cells from NOD DN cells appears intrinsic to these thymic-derived cells,two concerns could be raised.First,these cells might have already encountered biasing interactions in the thymus from which we isolated them.Second,the OP9-DL1cultures present the Delta-like 1(DL1)Notch ligand,whereas the Notch ligand ex-pressed in the normal thymus is Delta-like 4(DL4),which pro-
828b -AND gd -SELECTION CHECKPOINT ABNORMALITIES IN NOD MICE
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
vides a somewhat different Notch signal to these cells (39–41).Therefore,we also tested the T lineage development of pre-thymically derived BMC progenitors in vitro,providing DL4as the source of Notch ligands rather than DL1.As shown in Fig.2A ,cells derived from precursor-enriched B6and NOD BMCs gen-erate both DP and gd T cells on OP9-DL4after 19–22d of culture.However,NOD progenitor cells again exhibited a poor differen-tiation of DP cells (Fig.2A ,left panels )and enhancement of gd T cell generation (Fig.2A ,right panels )in comparison with those from B6mice.Furthermore,when NOD and B6cells were co-cultured,similar differences in ab T versus gd T development were observed between the two strains (Fig.2B ).As shown in two independent cultures,NOD cells (CD45.1+)produced a lower proportion of DP cells and more gd T cells than those of cultured cells from B6mice (CD45.2+).These results con?rm the profound
intrinsic and lineage-speci?c differences in T cell developmental programming between NOD and B6precursor cells.NOD DN2cells generate a higher absolute number of gd T cells and fewer DP cells than those generated by B6DN2cells in vitro
Skewed production of gd T cells relative to DP cells could result from lineage-speci?c alterations in proliferation or survival as well as lineage choice itself.To distinguish between these possi-bilities,we focused on the DN2cells,which particularly differed between NOD and B6in terms of their abilities to generate DP versus gd T cells in OP9-DL1cocultures.First,we assessed the total cell numbers of DP and gd T cells generated from limited numbers of precursor cells in these cultures.Ten DN2cells from B6and NOD thymi were sorted directly into each of 24
individual
FIGURE 1.NOD DN cells generate and maintain few DP ab T cells while retaining gd T development in vitro.A ,Flow cytometric analyses showing the development of DP and gd T cells from magnetic bead-puri?ed DN cells from B6,NOD,and BALB/c thymocytes after 12d of OP9-DL1coculture in 5ng/ml IL-7and Flt3L.Similar results were obtained from cultures harvested on day 8and when IL-7concentrations were dropped to 2ng/ml for the last 5d of culture or were maintained at 2ng/ml throughout the culture period (data not shown).B –E ,Development of FACS sorted DN1,DN2,DN3a,and DN3b (post b -selection)cells from B6and NOD thymi cultured in 2.5ng/ml IL-7and 5ng/ml Flt3L.B ,Flow cytometric plots showing CD4and CD8DP cell development at days 7and 14from each progenitor population.C ,Histograms showing levels of surface TCR b expression in the same cultures for B6and NOD cell populations at days 7and 14of culture.No data are shown for NOD DN3b cells on day 14because very few cells are remaining.D ,Flow cytometric plots of CD44versus CD25showing differences in initial DN differentiation in the same cultures at day 7.E ,gd T cell development in the same cultures at day 14.Percentages of cells in each gate are indicated.These results are representative of at least three independent experiments.
The Journal of Immunology 829
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
OP9-DL1cultures and analyzed by ?ow cytometry on day 13.The percentage of gd T cells out of the total number of differentiated cells (calculated as 1003number of gd T/[number of gd T +number of DP])for each well is shown in Fig.3A (left panel ),and a highly signi?cant difference was found between cultured B6and NOD DN2cells.Although DN2cells from NOD and B6thymi yielded comparable total numbers of CD45+cells per well,NOD cells produced signi?cantly higher absolute numbers of gd T cells,as well as signi?cantly fewer DP cells (Fig.3A ,right panels ).Thus,both poor DP cell differentiation and enhanced gd T cell development contribute to the NOD phenotype.
To determine whether the differences in ab T versus gd T dif-ferentiation are the result of alterations in lineage choice,single-cell differentiation analyses were performed.Because commitment to the gd T cell lineage may occur as early as the DN2stage (42),we sought to determine if DN2cells from either mouse strain start with a biased frequency of cells precommitted to either lineage.For this experiment,single cells from two subsets of the DN2CD44+CD25+c-Kit hi population were tested separately based on their surface c-Kit levels,the less mature DN2a (CD44+CD25+c-Kit ++)cells and the more mature,T lineage-committed DN2b (CD44+CD25+c-Kit +)cells (33).Each well was scored for the presence and numbers of gd T and DP cells after 13d of OP9-DL1culture.Results in Fig.3B (left panel )show again that NOD DN2cells,both DN2a and DN2b,generally produce proportionately more gd T cells and fewer DP cells as a percentage of total differentiated cells than those of corresponding cells from B6mice.However,differences between B6and NOD were clearly not due to pre-mature commitment in cells from either strain as they exhibited similar percentages of bipotent cells (black bars)at each stage (Fig.3B ,right panel ).Almost all DN2a cells from both strains produced both gd T and DP cells.Although the majority of DN2b cells were still bipotent,individual DN2b cells from both NOD and B6mice
were more likely than DN2a-stage cells to generate only DP cells,a trend that continued to the DN3a stage when few cells remained bipotent and most generated only DP cells,as shown previously for B6mice (34).Few single cells from either strain produced only gd T cells.Thus,the high proportion of gd T cells and low proportion of DP cells in NOD cultures is not a result of increased previous gd T lineage commitment of the precursors but rather the result of enhanced gd T cell production and reduced DP cell generation after the lineage choice is made.
NOD gd T cells proliferate more rapidly than do B6gd T cells in vitro
The normal preponderance of ab -lineage DP cells over gd T cells in the thymus depends not only on the frequency of different se-lection events but also on the greater extent of net proliferation that is normally associated with the ab -lineage program after b -selection.To address the question of whether some of the dif-ferences in ab Τversus gd T cell development are attributable to a NOD defect in proliferation,unsorted thymocytes (“All”)as well as sorted DN1/ETP,DN2,DN3a,and DN3b populations were stained with CFSE and placed in OP9-DL1culture.NOD DN subsets showed similar or slightly faster proliferation rates than B6cells over the ?rst 4d of culture (Fig.4A ),ruling out a generalized proliferation defect.Most prominently,the gd TCR-positive cells generated in the ?rst 4d from NOD DN3a cultures undergo more rapid and extensive proliferation than that of gd TCR-positive cells from B6cell cultures,whereas the gd TCR-negative cells from NOD and B6cells proliferated more similarly (Fig.4B ).Thus,the high numbers of gd T cells generated from NOD DN cells result,at least in part,from their greater proliferation after surface TCR gd expression in comparison with B6gd T cells.Furthermore,the lower numbers of DP cells generated from NOD DN3cells is not due to an initial defect in proliferation after b
-selection.
FIGURE 2.NOD bone marrow progenitor cells also show attenuated in vitro development of DP ab T cells and enhanced gd T cell development.BMCs enriched for progenitors were isolated from NOD and B6mice and cultured on OP9-DL1and DL4cells as described in Materials and Methods .Flow cytometry plots show the gener-ation of CD4/CD8DP cells and gd T cells from B6and NOD bone mar-row cultures analyzed after 19and 22d (A )and two independent cultures established from mixing equal num-bers of NOD and B6BMCs,analyzed after 20d (B ).Results are representa-tive of at least three replicate wells from one of two independent experi-ments with similar results.
830b -AND gd -SELECTION CHECKPOINT ABNORMALITIES IN NOD MICE
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
NOD and B6thymic gd T cells exhibit similar patterns of gene expression
We recently reported the expression of many key T cell genes in DN populations and gd T cells from NOD and B6mice and found no major differences in expression of Ptcra,Notch1,Hes1,Tcf7, Runx1,and Bcl11b among others(33).However,recent evidence suggests that gd T cells may be composed of multiple lineages, distinguished by their different dependence on speci?c transcrip-tion factors:Sox13,Id3,or Zbtb16/Plzf(37,43–45).To determine whether NOD thymocytes preferentially generate all classes of gd T cells or just one particular subtype,cells were analyzed for expression of these transcription factor genes as well as for diag-nostic TCR-V g rearrangements.We sampled V g rearrangements representing members of the TCR-C g1cluster,which includes adult,fetal,and tissue-speci?c receptors(reviewed in Ref.8).We also assayed for V g1.1-J g4rearrangements,which mark a re-cently characterized population of innate gd T cells with V g1.1-V d6.3TCR gd receptors.These cells rapidly produce IL-4and IFN-g,upregulate Zbtb16/PLZF,a critical transcription factor in NKT differentiation(45),and are Id3independent(37).NOD and B6gd T cells expressed fairly similar levels of transcripts from V g3,V g5,V g4,and V g2to C g1rearrangements,as well as V g1.1-J g4rearrangements(Fig.5).No differences were observed in expression of Id3or of the CD3e,CD247(CD3z),and
Sox13
FIGURE 3.NOD and B6DN2 cells generate more gd T and fewer DP cells in10-cell and single-cell OP9-DL1cocultures but retain bipo-tency.A,Plots showing the relative proportion of gd T cells in compari-son with DP cells generated from each individual B6and NOD10-cell DN2cell culture,calculated as%gd T/(gd T+DP).The absolute numbers of CD45+total cells,gd T cells,and DP cells generated in each of those cultures are also shown.A horizontal bar indicates the median value of in-dividual B6or NOD cultures.B,Plot showing the relative proportions of gd T cells in comparison with DP (calculated as%gd T/[gd T+DP]) generated from single-cell cultures of sorted DN2a,DN2b,and DN3a cells (left).To determine the commitment of the cells to ab versus gd lineages, the lineage choices of individual cells are shown as percentages of indi-vidual wells containing gd T cells only,both gd T and DP cells,or DP cells only(right).The p values given below the plots were determined us-ing Mann–Whitney U tests.Data are representative of two independent ex-
periments.
FIGURE4.B6and NOD DN populations proliferate
at similar rates in vitro,but NOD gd T cells proliferate
more rapidly than B6gd T cells.DN cell populations
were puri?ed by cell sorting and stained with CFSE
before coculture with OP9-DL1cells.A,Total thymo-
cytes(All)and puri?ed DN1,DN2,DN3a,and DN3b
cells were harvested for analysis of remaining CFSE
levels on days1,2,3,and4.B,Sorted DN3a cells were
cultured on OP9-DL1cells,harvested after4d,and
stained to distinguish TCR gd+cells.Histograms show
CFSE levels for B6(solid gray)and NOD(black line)
cells.Data are representative of three independent ex-
periments with similar results.
The Journal of Immunology831
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/
Downloaded from
genes.However,Zbtb16/Plzf expression was somewhat higher in NOD gd T cells in comparison with B6cells.Given the similarity in TCR V g usage,this could re?ect a generally elevated state of activation (45).Thus,there appear to be few if any major differ-ences in the types of gd T cells produced in NOD and B6mouse thymi,suggesting that the differences we observe in develop-ment and rates of proliferation might be generalized to multiple gd T cell subsets.
NOD thymocytes exhibit multiple abnormalities around the b -selection checkpoint
A number of costimulatory and other cell surface receptors,with known effects on TCR signals,survival,and/or proliferation,are induced at b -and gd -selection in response to TCR signaling via the newly produced pre-TCR and gd TCR,similar to events after the activation of peripheral T cells.The combinations and levels of these molecules can not only indicate the nature and intensity of the TCR signals but also in?uence further response thresholds through their modulating and enhancing roles.The degree of CD5upregulation,for example,has been a useful indicator of the in-tensity of signals driving gd -selection versus b -selection (10),and once induced,CD5can affect further development (10)and signal thresholds for positive and negative selection (46,47).
We have previously shown that upregulation of the costimu-latory receptor,CD27,involved in mature T cell survival and proliferation,can be used to distinguish DN3cells that have re-arranged and transcribed an intracellular (ic)TCR b (DN3b)from those that are still TCR-negative in B6mice (34).However,NOD DN3cells express lower amounts of CD27after b -selection (icTCR b +)(Fig.6A ,top row ,left panel ).The costimulatory re-ceptor CD28,which is not expressed until after b -(or gd -)se-lection (48),and CD5are also induced to a lower level in TCR b +NOD DN cells than that in B6cells (Fig.6A ,top row ,second and third panels ).These observed differences are not due to variations in the total amount of TCR b expressed nor to cell size,as the icTCR b staining intensity and FSC histograms are similar be-tween the two strains (Fig.6A ,top row ,fourth and ?fth panels ).In contrast to the results for ab T cell precursors,icTCR gd +cells from B6and NOD thymi express similar levels of surface CD27and CD28(Fig.6A ,bottom row ,?rst and second panels ).Furthermore,NOD gd T cells express a broader range of surface CD5levels,both higher and lower,and a higher total amount of TCR gd than that expressed by B6gd T cells,despite comparable cell sizes as determined by FSC pro?le (Fig.6A ,bottom row ,fourth and ?fth panels ).The higher levels of CD27and CD5
attained by thymic gd T cells from both NOD and B6mice re?ect the higher intensity of signals generated at gd -selection compared with those generated at b -selection (9,10).Likewise,the lower induction of CD27,CD28,and CD5in icTCR b +cells from NOD mice compared with that in icTCR b +cells from B6mice suggests that NOD cells may experience a weaker or otherwise altered b -selection signal,whereas gd T cell signals are more similar between the two strains.
Depletion of subsets after b -selection in the normal NOD thymus
B6and NOD thymi exhibit few obvious differences in their pro-portions of major populations in steady state (17,18).However,because the OP9-DL1coculture system revealed major intrinsic defects in NOD differentiation at the earliest TCR-dependent checkpoint,we performed a series of detailed ?ow cytometric analyses of freshly puri?ed NOD and B6thymocytes to evaluate more closely if DN cells within the NOD thymus exhibit any comparable abnormalities in vivo.We found that whereas all DN populations are present,as measured by CD44and CD25pro?les,the percentages and shapes of the differentiation plots differ (Fig.6B ,left panels ).Most prominently,the levels of surface CD25are much higher in CD25+cells from NOD than in B6cells,as seen in the density plots,as well as in histograms of all CD44lo cells (including DN3to DN4cells)(Fig.6B ,right panel ).Furthermore,NOD thymi consistently have a lower proportion of DN4cells relative to DN3cells (Fig.6B ,left panels ,lower left versus lower right quadrants ).Elevated CD25in DN3cells and lower pro-portions of DN4cells can be indicative of a partial block in b -selection as reported for thymi from mice with mutations in the pre-TCR or Notch signaling pathways (49–52).
The generally accepted route of differentiation for B6mouse ab T cells,after b -selection and the downregulation of CD25leading to the DN4stage,is the upregulation of CD8into immature single-positive (ISP)cells prior to the expression of surface CD4in the DP stage (7).A previous study reported low numbers of CD8+ISP cells in NOD thymi (17).To con?rm this ?nding and to determine the route of differentiation from DN4to DP in NOD thymi,CD8ISP cells were distinguished from mature CD8single-positive (SP)cells by their retention of high levels of surface CD24(heat shock Ag;HSA),which then decline during differentiation.B6thymocytes follow this pattern,expressing a high percentage of total gated CD8+cells that are HSA hi and CD2int (ISP)cells compared with that of the mature HSA intermediate and CD2hi SP CD8+cells (Fig.6C ).However,NOD thymocytes have a much
FIGURE 5.Expression of TCR-V g rear-rangements and other genes in sorted thymic NOD and B6gd T cells.Quantitative PCR results from TCR gd +cells sorted from NOD and B6thymi using primers to detect subsets of speci?c TCR-V g rearrangements and other T cell genes.Data are shown as the geometric mean 6SD,n =2–5.For additional gene expression comparisons,see Ref.33.
832b -AND gd -SELECTION CHECKPOINT ABNORMALITIES IN NOD MICE
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
lower percentage of CD8ISP cells that is only partially offset by a corresponding HSA hi CD2int CD4+ISP population.Furthermore,none of the populations from NOD thymi have a major subset of cells with the same level of HSA that is expressed by the B6ISP cells,as indicated by the red boxes (Fig.6C ,right panels ),sug-gesting a paucity of cells transiting through this stage of de-velopment.
The interpretation that there might be a reduced in?ux into the ISP compartment from previous stages was further supported by the kinetics of proliferation and turnover of DN and DP subsets in B6and NOD thymi in vivo,as determined by the fate of a cohort of BrdU pulse-labeled cells.A previous study reported an anomalous dip in BrdU +DP cells in NOD mice after 2–3d (18).We wished to con?rm and extend this ?nding in the context of a b -selection defect by additional DN cell analysis.Mice were injected i.p.with BrdU,sacri?ced after 1,2,or 3d,and the thymocytes were stained for surface receptors and intracellular BrdU.One day after BrdU injection,the proportions of labeled cells within the DN2/DN3(CD25hi ),DN4(CD252CD442),and DP populations were simi-lar between NOD and B6(Fig.6D ).The DN2/3cells were ex-pected to accumulate BrdU +cells due to extensive proliferation at the DN1and DN2stages,although most DN3cells normally pause in proliferation before b -selection.More than 50%of the DN4cells were pulse-labeled at this early time point,because cells that have passed through the b -selection checkpoint from DN3undergo a rapid burst of proliferation while differentiating (Fig.4A ,DN3b cells).DP cells proliferate very little,but BrdU +cells are expected to ?ow into the DP compartment and accu-mulate during subsequent days due to the continuing differentia-tion of the rapidly proliferating DN4and ISP cells.Accordingly,on day 2postinjection,the percentage of BrdU +DN4cells from both B6and NOD declined precipitously (Fig.6D ,middle panel
),
FIGURE 6.NOD thymocytes show evidence of a partial block at b -selection and abnormalities in differentiation to DP cells in vivo.A ,Histograms showing levels of activation-induced surface receptors CD27,CD28,and CD5on freshly isolated DN thymocytes from NOD (black lines)and B6(solid gray)mice,using intracellular (ic)staining for TCR b (top row )or TCR gd (bottom row )to distinguish cells that have passed through b -selection or gd -selection,respectively.Data are representative of three to four independent experiments.B ,Flow cytometric analysis showing CD44versus CD25expression in DN subsets from freshly isolated NOD and B6thymi (left panels ).Percentages of cells in each quadrant are as indicated.The histogram compares CD25surface expression levels between gated NOD and B6CD44lo DN cells (DN3and DN4cells)from two mice of each strain (right panel ).Data are representative of .10mice of each strain.C ,Flow cytometric analyses showing CD4versus CD8plots for total thymocytes from NOD and B6mice (left panels ),CD8and CD4gated cells,stained to distinguish immature (ISP)cells from mature SP cells (middle panels ),and histograms showing HSA levels for DN,DP,CD8+,and CD4+cells (right panels ),which distinguish less mature cells (HSA hi )from more mature cells (HSA lo ).The red boxes indicate the intermediate HSA level for B6CD8ISP cells (right top panel ,arrow)with no corresponding major population in the NOD thymus (right bottom panel ).Data are representative of three independent experiments.D ,Percentages of BrdU +cells among DN2/3(CD25+),DN4(CD252),and DP cell populations 1–3d after in vivo BrdU pulse-labeling,showing a major difference in accumulation of BrdU +DP cells on day 2between B6and NOD mice.Mean 6SD values over time are plotted for B6and NOD thymocytes.
The Journal of Immunology 833
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
as expected due to turnover and differentiation from nonlabeled DN3precursors,con?rming the pulse nature of the labeling.The B6DP compartment showed an almost 2-fold increase in the proportion of BrdU +cells over the proportion at day 1,again as expected due to continuing differentiation of the BrdU +-labeled DN4cells from the preceding days.However,the NOD DP com-partment did not show a corresponding increase in the percentage of BrdU +cells at day 2,but rather showed a decline.The failure of BrdU-labeled NOD cells to become DP was not due to their re-tention in the DN4compartment.The rapid loss of BrdU +cells from all phenotypic compartments suggests that failure to accu-mulate ISP cells in NOD thymi is not due to poor proliferation or rapid modulation of surface CD4,CD8,and HSA,but instead due to a survival failure.Taken together,our results support the notion that NOD early T cells in vitro and in vivo have a partial block around the b -selection checkpoint,leading to a reduction in DP cell accumulation.
Lineage-speci?c differences in the establishment of ab T
versus gd T cell and B cell populations in mixed bone marrow chimeras
To determine whether preferential development of gd T cells,as well as reduced production of ab T cells,from NOD precursors occur in vivo as well as in vitro,we used mixed bone marrow chi-meras to compare the kinetics of T cell differentiation and export between NOD and B6cells in the same animal.An equal number of NOD and B6BMCs were injected into irradiated (NOD 3B6)F1.Rag12/2mice,which were assayed for reconstitution at 5–9
wk of age.Five weeks postinjection,both NOD (CD45.1+)and B6(CD45.2+)cells were found in the thymus (Fig.7A ,left panels )with relatively normal steady-state proportions of DN,DP,SP,and gd T populations established at this time.However,a lower pro-portion of CD45.1+NOD cells was found in the spleen compared with that of CD45.2+B6cells (Fig.7A ,right panels ).The NOD cells were predominately CD19+B cells with a T/B cell ratio of 2.2:85compared with the B6T/B ratio of 35:56(Fig.7A ,right top panels ).This T cell repopulation defect was primarily due to dis-proportionately fewer TCR ab +T cells.In contrast,relatively normal percentages and total yields of NOD TCR gd cells were found.Thus,in vivo as well as in vitro,there is an NOD defect in production of ab T cells,and this is lineage speci?c.
A summary of results from all six mixed bone marrow chimeric mice analyzed at 5–6and 8–9wk of age is shown in Fig.7
B and 7
C .In 5of 6thymi and 6of 6spleens from chimeric mice,NO
D cells exhibited a lower ratio of TCR ab +/TCR gd +cells compared with that of B6cells in the same mouse.In agreement with the steady-state phenotype of NOD mice,this effect was kinetic,not absolute.At the later time point,by 8–9wk postinjection (Fig.7B ,4and 5),the relative proportions of TCR ab +/TCR gd +cells in the thymus and spleen became more similar between NOD and B6than those observed at 5–6wk (Fig.7B ,1–3),indicating that NOD ab T cells eventually accumulate.The defect in NOD ab T production was again shown to be lineage speci?c,as relatively high numbers of B cells were produced in these animals (Fig.7C ).Thus,NOD BMCs were much less ef?cient than B6BMCs at producing mature ab T cells in chimeric hosts,even though they were capable of rapidly generating gd T and B cells.These
results
FIGURE 7.NOD cells exhibit differences in establishment of gd T versus ab T cells in mixed bone marrow chimeras.BMCs isolated from NOD and B6mice and 53106cells from each strain were mixed and injected into irradiated (NOD 3B6)F1.Rag12/2recipients.Mice were sacri?ced after 5–6or 8–9wk and analyzed by ?ow cytometry for lymphocyte populations in thymus and spleen using CD45.1(NOD)and CD45.2(B6)to distinguish donor strains.A ,Flow cytometric analysis of a representative chimeric mouse (No.1)analyzed at 5wk postinjection.Plots on the left show thymocytes were stained for CD4and CD8(upper panels )and CD3εand TCR gd (lower panels ).Splenocytes,shown on the right ,were stained for ab TCR and CD19,to distinguish B cells (upper panels ),and CD3εand TCR gd (lower panels ).B and C ,Summary of FACS staining results from six mixed bone marrow chimeric mice,three analyzed after 5–6wk (No.1–3)and three after 8–9wk (No.4–6).B ,Graphs showing the ratio of TCR ab +/TCR gd +cells in thymus (left )and spleen (right )generated from B6(black bars)and NOD (white bars)donors.C ,Graph showing the relative percentages of B,ab T,and gd T lymphocytes derived from B6and NOD donors in spleens from the same individual chimeric mice.
834b -AND gd -SELECTION CHECKPOINT ABNORMALITIES IN NOD MICE
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
show that the NOD genotype confers divergent effects on ab and gd lineage differentiation in vivo as well as in vitro.
Discussion
The ?rst TCR-dependent checkpoint is the ?rst of many stages in which the TCR signaling apparatus of the cell is used for survival,lineage choice,and differentiation functions.The earliest T lineage decision,which is made at this point,is the fundamental choice between ab T and gd T cell fates,which is determined by dif-ferences in TCR signal strength and results in initiation of very different developmental programs.Furthermore,recent evidence suggests that different gd T cell lineages may be selected at this point depending on the characteristics of the rearranged TCR and transduced signal at this stage (reviewed in Ref.43).
The OP9-DL1coculture system provides the Notch ligands and other factors required by early T cells for passage through the ?rst TCR-dependent checkpoint and differentiation into gd Τor ab T cells,and it is a powerful tool allowing detailed study of the T cell developmental program and lineage choice in both mice and humans (29–32,53,54).Using this in vitro culture system,we show that NOD early DN cells are very poor at generating DP ab T cells,but the same cells under the same conditions exhibit en-hanced gd T cell differentiation and proliferation.This was true of puri?ed DN thymocytes as well as precursor enriched BMCs and in the presence of DL4,the normal thymic Notch ligand,rather than DL1.Because the DN thymocytes are the progenitors of all T cell lineages,defects expressed at these earliest stages may also be present in all subsequent stages and may have a major impact on later thymic selection events,lineage choices,and peripheral developmental choices and responsiveness.
Although the OP9-DL1cell culture system may exacerbate the relatively poor DP differentiation by NOD early T cell progenitors,we have several additional lines of evidence that indicate that b -selection may also be defective in the NOD thymus.First,the heightened CD25expression in NOD DN3cells is similar to pre-TCR and Notch mutations that give a partial block at b -selection (49–52).Second,CD5can be used as an indicator of the intensity of TCR signaling (10),and b -selected icTCR b +cells from NOD thymi weakly upregulate coreceptor molecules CD5,as well as CD27and CD28,compared with that of the corresponding pop-ulations from B6thymi.The low proportion of CD8+ISP cells found in NOD thymi has been previously reported (17),but the paucity of HSA-intermediate cells in any corresponding pop-ulation,and the poor accumulation of DP cells in BrdU-labeling studies,suggest that this may be due to an impairment of the cells differentiating past b -selection to the DP compartment rather than simply use of an alternate route of differentiation.The difference does not appear to be at the level of cell proliferation after b -se-lection as DN3b cells from NOD thymi proliferate at a rate similar to that of B6DN3b cells,as measured by CFSE.CD27is asso-ciated with proliferation and survival of cells after b -selection,as well as survival of peripheral CD8T cells (55,56),and it is possible that the low level of CD27upregulation upon b -selection in NOD mice may contribute to the poor accumulation of DP cells in the BrdU experiments and in OP9-DL1culture.Overall,these results are consistent with previous reports showing that DP cells accumulate very slowly in NOD thymi after depletion of the DP compartment with dexamethasone (20)and can be interpreted as an impairment in b -selection in NOD mice.
The preferential development of gd T cells from NOD DN subsets is not simply a concentration artifact resulting from the poor DP differentiation in these animals.There is evidence that some gd T cell precursors can become committed precociously at the DN2stage (42,57),before cells reach the conventional b -
selection checkpoint.That does not appear to be the case here,as single DN2cells sorted from NOD thymi were predominately bipotent just as in B6thymi,despite producing relatively higher numbers of gd T cells compared with that of DP cells.Thus,the skewing of NOD differentiation toward the gd T lineage is more likely to be due to survival and proliferation than to lineage choice.The major differences in ab T versus gd T cell develop-ment in vitro may result from alterations in intrinsic signaling pathways in NOD early T cells with differential effects on the two lineages.For example,stronger intrinsic IL-7signals,lower Notch signals,weaker b -catenin/TCF-1activity,and stronger TCR sig-nals have all been shown to favor the differentiation,survival,and/or proliferation of gd T cells over ab T cells (58–62).Although lineage choice may be normal in NOD DN cells,their ability to survive,proliferate,and differentiate in the chosen lineage may differ between b -and gd -selected T cells.Normally,in B6and most strains examined,the b -selection program dictates much greater proliferative expansion than that of gd -selection.Our re-sults imply that this difference is greatly reduced or reversed in NOD mice.
In addition to their preferential development from NOD DN cells in OP9-DL1culture,we found that gd T cells generated in vivo in NOD thymi proliferate more,express a wider range of surface CD5,and upregulate all assayed activation-induced receptors very well.This suggests that NOD gd T cells may be produced as a result of unusually robust or variable signaling responses in the thymus.Thus,it is possible to interpret the preferential devel-opment of gd T cells from NOD precursors as an outcome from a stronger or altered TCR signal,such that gd T cell differentia-tion,proliferation,and survival is actively promoted (9,10,63,64)whereas DP differentiation and survival are disfavored (54).Al-ternatively,the TCR signal may not be stronger in NOD cells,but they may interpret any viability-promoting signal as a gd T cell selection signal,thereby leaving many cells that undergo the b -selection program starved of viability support.Although further work is needed to resolve which intrinsic mechanisms are af-fecting ab versus gd T cell differentiation in NOD DN cells,there is evidence that immature and mature NOD T cells may transduce abnormal TCR signals during activation (reviewed in Ref.12).In addition,NOD.Prkdc scid /scid and NOD.Rag 2/2thymocytes fail to arrest at the b -selection checkpoint,undergoing spontaneous dif-ferentiation to DP cells without restoration of thymic cellularity (19),suggesting that NOD DN cells,even in the absence of a rearranged TCR,may undergo a spontaneous,but only partial,TCR signal.A number of factors can cause this type of break-through phenotype including enhanced TCR and Notch signals (6,65).Notably,we previously reported data showing a higher level of Notch3and Dtx1expression,but not Notch1or Hes1,in freshly isolated NOD DN3a cells in comparison with that of B6DN3a cells (33),although the signi?cance of this difference is not yet clear.Recent studies have demonstrated that events at b -selection or even earlier can have a major effect on the characteristics and further lineage choices of ab TCR +DP cells (66–68).Loss of a subset of differentiating cells after b -selection might result in a differentially selected DP population that,for example,trans-duces a stronger than average TCR signal or is more resistant to certain types of apoptosis.If aberrant NOD b -selection events alter the signaling characteristics of the ?nal DP population,this could have major rami?cations for negative and positive selection of conventional CD4and CD8T cells.The same could be true for the selection of gd T cells as well as “unconventional”ab T cells with self-reactive TCRs,such as NKT,CD8aa ,and regulatory T cells (69).In addition,although little is known about possible roles of gd T cells in autoimmunity,gd T intraepithelial lym-
The Journal of Immunology
835
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
phocytes may play an important role in peripheral tolerance and have been found to be able to control diabetes progression in NOD mice (14).gd T cells reactive to insulin peptide B:9-23have been reported to be generated in NOD but not B6mice (70),which provides further evidence of their potential role in autoimmune diabetes.The diversity of gd T lineages,their peripheral immune responses,and their roles in disease have been a subject of great interest very recently,especially in light of the ability of particular subsets to rapidly secrete IL-17,IFN-g ,and IL-4(reviewed in Refs.43,71).
The results of this study show that NOD lineage T cells have fundamental and intrinsic abnormalities from the earliest stages in their responses to activating signals,either via TCR or other en-vironmental triggers,even in the absence of MHC/peptide/APC interactions.These defects may affect lineage choices and TCR repertoires via alterations in DP selection thresholds or alter pe-ripheral immune responses to speci?c stimuli,or both.Finally,whether or not these traits found in NOD early T development directly contribute to autoimmunity,these studies demonstrate that different mouse strains can show major variations in b -and gd -selection developmental programming,which may deviate sig-ni?cantly from the currently accepted pathways.
Acknowledgments
We thank J.C.Zun ?iga-P?u ¨cker for generously providing OP9-DL1and
OP9-DL4cells for these experiments,Rochelle Diamond,Stephanie Adams,Diane Perez,and Pat Koen from the Caltech Cell Sorting Facility for cell sorting,Natasha Bouey,Robert Butler,Ruben Bayon,and Beth Olsen for excellent animal care,Marion Duprilot for preliminary gene ex-pression data,and Tom Taghon (University of Ghent,Belgium)and mem-bers of the Rothenberg laboratory,especially Rochelle Diamond,for helpful suggestions.
Disclosures
The authors have no ?nancial con?icts of interest.
References
1.Rothenberg,E.V .,J.E.Moore,and https://www.doczj.com/doc/794309662.html,unching the T-cell-lineage developmental programme.Nat.Rev.Immunol.8:9–21.
2.Godfrey,D.I.,J.Kennedy,T.Suda,and A.Zlotnik.199
3.A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8-triple-negative adult mouse thymocytes de?ned by CD44and CD25expression.J.Immunol.150:4244–4252.
3.Ceredig,R.,and T.Rolink.2002.A positive look at double-negative thymocytes.Nat.Rev.Immunol.2:888–897.
4.von Boehmer,H.,I.Aifantis,J.Feinberg,O.Lechner,C.Saint-Ruf,U.Walter,J.Buer,and O.Azogui.1999.Pleiotropic changes controlled by the pre-T-cell receptor.Curr.Opin.Immunol.11:135–142.
5.Michie,A.M.,and J.C.Zu ′n ?iga-P?u ¨cker.2002.Regulation of thymocyte dif-ferentiation:pre-TCR signals and beta-selection.Semin.Immunol.14:311–323.
6.Kruisbeek,A.M.,M.C.Haks,M.Carleton,A.M.Michie,J.C.Zu ′n ?iga-P?u ¨cker,
and D.L.Wiest.2000.Branching out to gain control:how the pre-TCR is linked to multiple functions.Immunol.Today 21:637–644.
7.Rothenberg,E.V .,and M.A.Yui.2008.Development of T cells.In Fundamental Immunology ,6th Ed.W.E.Paul,ed.Lippincott Williams &Wilkins,Phila-delphia,PA,p.376–406.
8.Xiong,N.,and D.H.Raulet.2007.Development and selection of gammadelta T cells.Immunol.Rev.215:15–31.
9.Haks,M. C.,J.M.Lefebvre,https://www.doczj.com/doc/794309662.html,uritsen,M.Carleton,M.Rhodes,T.Miyazaki,D.J.Kappes,and D.L.Wiest.2005.Attenuation of gamma-deltaTCR signaling ef?ciently diverts thymocytes to the alphabeta lineage.Im-munity 22:595–606.
10.Hayes,S.M.,L.Li,and P.E.Love.2005.TCR signal strength in?uences
alphabeta/gammadelta lineage fate.Immunity 22:583–593.
11.Anderson,M.S.,and J.A.Bluestone.2005.The NOD mouse:a model of im-mune dysregulation.Annu.Rev.Immunol.23:447–485.
12.Delovitch,T.L.,and B.Singh.1997.The nonobese diabetic mouse as a model of
autoimmune diabetes:immune dysregulation gets the NOD.Immunity 7:727–738.
13.Salomon,B.,D.J.Lenschow,L.Rhee,N.Ashourian,B.Singh,A.Sharpe,and
J.A.Bluestone.2000.B7/CD28costimulation is essential for the homeostasis of the CD4+CD25+immunoregulatory T cells that control autoimmune diabetes.Immunity 12:431–440.
14.Locke,N.R.,S.Stankovic,D.P.Funda,and L.C.Harrison.2006.TCR g d
intraepithelial lymphocytes are required for self-tolerance.J.Immunol.176:6553–6559.
15.Wicker,L.S.,https://www.doczj.com/doc/794309662.html,ler,and Y .Mullen.1986.Transfer of autoimmune di-abetes mellitus with splenocytes from nonobese diabetic (NOD)mice.Diabetes 35:855–860.
16.Bendelac,A.,C.Carnaud,C.Boitard,and J.F.Bach.1987.Syngeneic transfer of
autoimmune diabetes from diabetic NOD mice to healthy neonates.Requirement for both L3T4+and Lyt-2+T cells.J.Exp.Med.166:823–832.
17.Godfrey,D.I.,S.J.Kinder,P.Silvera,and A.G.Baxter.1997.Flow cytometric
study of T cell development in NOD mice reveals a de?ciency in alphabetaTCR +CDR-CD8-thymocytes.J.Autoimmun.10:279–285.
18.Berzins,S.P.,E.S.Venanzi,C.Benoist,and D.Mathis.2003.T-cell compart-ments of prediabetic NOD mice.Diabetes 52:327–334.
19.Yui,M.A.,and E.V .Rothenberg.2004.Deranged early T cell development in
immunode?cient strains of nonobese diabetic mice.J.Immunol.173:5381–5391.
20.Bergman,M.L.,C.Penha-Gonc ?alves,K.Lejon,and D.Holmberg.2001.Low
rate of proliferation in immature thymocytes of the non-obese diabetic mouse maps to the Idd6diabetes susceptibility region.Diabetologia 44:1054–1061.21.Zipris,D.,https://www.doczj.com/doc/794309662.html,zarus,A.R.Crow,M.Hadzija,and T.L.Delovitch.1991.
Defective thymic T cell activation by concanavalin A and anti-CD3in autoim-mune nonobese diabetic mice.Evidence for thymic T cell anergy that correlates with the onset of insulitis.J.Immunol.146:3763–3771.
22.Kwon,H.,H.S.Jun,Y .Yang, C.Mora,S.Mariathasan,P.S.Ohashi,
R.A.Flavell,and J.W.Yoon.2005.Development of autoreactive diabetogenic T cells in the thymus of NOD mice.J.Autoimmun.24:11–23.
23.Kishimoto,H.,and J.Sprent.2001.A defect in central tolerance in NOD mice.
Nat.Immunol.2:1025–1031.
24.Lesage,S.,S. B.Hartley,S.Akkaraju,J.Wilson,M.Townsend,and
C.C.Goodnow.2002.Failure to censor forbidden clones of CD4T cells in autoimmune diabetes.J.Exp.Med.196:1175–1188.
25.Liston,A.,S.Lesage,D.H.Gray,L.A.O’Reilly,A.Strasser,A.M.Fahrer,
R.L.Boyd,J.Wilson,A.G.Baxter,E.M.Gallo,et al.2004.Generalized re-sistance to thymic deletion in the NOD mouse;a polygenic trait characterized by defective induction of Bim.Immunity 21:817–830.
26.Holler,P.D.,T.Yamagata,W.Jiang,M.Feuerer,C.Benoist,and D.Mathis.
2007.The same genomic region conditions clonal deletion and clonal deviation to the CD8alphaalpha and regulatory T cell lineages in NOD versus C57BL/https://www.doczj.com/doc/794309662.html,A 104:7187–7192.
27.Wagner,M.J.,S.Hussain,M.Mehan,J.M.Verdi,and T.L.Delovitch.2005.A
defect in lineage fate decision during fetal thymic invariant NKT cell de-velopment may regulate susceptibility to type 1diabetes.J.Immunol.174:6764–6771.
28.Starr,T.K.,S.C.Jameson,and K.A.Hogquist.2003.Positive and negative
selection of T cells.Annu.Rev.Immunol.21:139–176.
29.Schmitt,T.M.,R.F.de Pooter,M.A.Gronski,S.K.Cho,P.S.Ohashi,and
J.C.Zu ′n ?iga-P?u ¨cker.2004.Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.Nat.Immunol.5:410–417.
30.Schmitt,T.M.,and J.C.Zu ′n ?iga-P?u ¨cker.2002.Induction of T cell development
from hematopoietic progenitor cells by delta-like-1in vitro.Immunity 17:749–756.
31.De Smedt,M.,I.Hoebeke,and J.Plum.2004.Human bone marrow CD34+
progenitor cells mature to T cells on OP9-DL1stromal cell line without thymus microenvironment.Blood Cells Mol.Dis.33:227–232.
32.Rothenberg,E.V .,and T.Taghon.2005.Molecular genetics of T cell de-velopment.Annu.Rev.Immunol.23:601–649.
33.Yui,M.A.,N.Feng,and E.V .Rothenberg.2010.Fine-scale staging of T cell
lineage commitment in adult mouse thymus.J.Immunol.185:284–293.
34.Taghon,T.,M.A.Yui,R.Pant,R.A.Diamond,and E.V .Rothenberg.2006.
Developmental and molecular characterization of emerging b -and gammadelta-selected pre-T cells in the adult mouse thymus.Immunity 24:53–64.
35.Taghon,T.,M.A.Yui,and E.V .Rothenberg.2007.Mast cell lineage diversion
of T lineage precursors by the essential T cell transcription factor GATA-3.Nat.Immunol.8:845–855.
36.Li,L.,M.Leid,and E.V .Rothenberg.2010.An early T cell lineage commitment
checkpoint dependent on the transcription factor Bcl11b.Science 329:89–93.37.Verykokakis,M.,M. D.Boos, A.Bendelac, E.J.Adams,P.Pereira,and
B.L.Kee.2010.Inhibitor of DNA binding 3limits development of murine slam-associated adaptor protein-dependent “innate”gammadelta T cells.PLoS ONE 5:e9303.
38.Hoffman,E.S.,L.Passoni,T.Crompton,T.M.Leu,D.G.Schatz,A.Koff,
M.J.Owen,and A.C.Hayday.1996.Productive T-cell receptor b -chain gene rearrangement:coincident regulation of cell cycle and clonality during de-velopment in vivo.Genes Dev.10:948–962.
39.Koch,U.,E.Fiorini,R.Benedito,V .Besseyrias,K.Schuster-Gossler,M.Pierres,
N.R.Manley,A.Duarte,H.R.Macdonald,and F.Radtke.2008.Delta-like 4is the essential,nonredundant ligand for Notch1during thymic T cell lineage commitment.J.Exp.Med.205:2515–2523.
40.Hozumi,K.,C.Mailhos,N.Negishi,K.Hirano,T.Yahata,K.Ando,S.Zuklys,
G.A.Holla ¨nder,D.T.Shima,and S.Habu.2008.Delta-like 4is indispensable in thymic environment speci?c for T cell development.J.Exp.Med.205:2507–2513.
41.Besseyrias,V .,E.Fiorini,L.J.Strobl,U.Zimber-Strobl,A.Dumortier,U.Koch,
M.L.Arcangeli,S.Ezine,H.R.Macdonald,and F.Radtke.2007.Hierarchy of
836b -AND gd -SELECTION CHECKPOINT ABNORMALITIES IN NOD MICE
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from
Notch-Delta interactions promoting T cell lineage commitment and maturation.J.Exp.Med.204:331–343.
42.Kang,J.,A.Volkmann,and D.H.Raulet.2001.Evidence that gammadelta versus alphabeta T cell fate determination is initiated independently of T cell receptor signaling.J.Exp.Med.193:689–698.
43.Kreslavsky,T.,M.Gleimer,and H.von Boehmer.2010.Alphabeta versus gammadelta lineage choice at the ?rst TCR-controlled checkpoint.Curr.Opin.Immunol.22:185–192.
44.
Melichar,H.J.,K.Narayan,S.D.Der,Y .Hiraoka,N.Gardiol,G.Jeannet,W.Held,C.A.Chambers,and J.Kang.2007.Regulation of gammadelta versus alphabeta T lymphocyte differentiation by the transcription factor SOX13.Sci-ence 315:230–233.
45.
Kreslavsky,T.,A.K.Savage,R.Hobbs,F.Gounari,R.Bronson,P.Pereira,P.P.Pandol?,A.Bendelac,and H.von Boehmer.2009.TCR-inducible PLZF transcription factor required for innate phenotype of a subset of gammadelta T cells with restricted TCR https://www.doczj.com/doc/794309662.html,A 106:12453–12458.
46.Azzam,H.S.,J. B.DeJarnette,K.Huang,R.Emmons, C.S.Park,C.L.Sommers,D.El-Khoury,E.W.Shores,and P.E.Love.2001.Fine tuning of TCR signaling by CD5.J.Immunol.166:5464–5472.
47.Tarakhovsky, A.,S. B.Kanner,J.Hombach,J. A.Ledbetter,W.Mu ¨ller,N.Killeen,and K.Rajewsky.1995.A role for CD5in TCR-mediated signal transduction and thymocyte selection.Science 269:535–537.
48.
Williams,J.A.,K.S.Hathcock,D.Klug,Y .Harada,B.Choudhury,J.P.Allison,R.Abe,and R.J.Hodes.2005.Regulated costimulation in the thymus is critical for T cell development:dysregulated CD28costimulation can bypass the pre-TCR checkpoint.J.Immunol.175:4199–4207.
49.Wolfer,A.,A.Wilson,M.Nemir,H.R.MacDonald,and F.Radtke.2002.In-activation of Notch1impairs VDJbeta rearrangement and allows pre-TCR-independent survival of early a b lineage thymocytes.Immunity 16:869–879.50.Zhang,W.,C.L.Sommers,D.N.Burshtyn,C.C.Stebbins,J.B.DeJarnette,R.P.Trible,A.Grinberg,H.C.Tsay,H.M.Jacobs,C.M.Kessler,et al.1999.Essential role of LAT in T cell development.Immunity 10:323–332.
51.Hinton,H.J., D.R.Alessi,and D. A.Cantrell.2004.The serine kinase phosphoinositide-dependent kinase 1(PDK1)regulates T cell development.Nat.Immunol.5:539–545.
52.
Mao,C.,E.G.Tili,M.Dose,M.C.Haks,S.E.Bear,I.Maroulakou,K.Horie,G.A.Gaitanaris,V .Fidanza,T.Ludwig,et al.2007.Unequal contribution of Akt isoforms in the double-negative to double-positive thymocyte transition.J.Immunol.178:5443–5453.
53.de Pooter,R.F.,and J.C.Zu ′n ?iga-P?u ¨cker.2007.Generation of immunocom-petent T cells from embryonic stem cells.Methods Mol.Biol.380:73–81.
54.Kreslavsky,T.,A.I.Garbe,A.Krueger,and H.von Boehmer.2008.T cell receptor-instructed alphabeta versus gammadelta lineage commitment revealed by single-cell analysis.J.Exp.Med.205:1173–1186.
55.
Gravestein,L.A.,W.van Ewijk,F.Ossendorp,and J.Borst.1996.CD27cooperates with the pre-T cell receptor in the regulation of murine T cell de-velopment.J.Exp.Med.184:675–685.
56.Hendriks,J.,Y .Xiao,J.W.Rossen,K.F.van der Sluijs,K.Sugamura,N.Ishii,
and J.Borst.2005.During viral infection of the respiratory tract,CD27,4-1BB,and OX40collectively determine formation of CD8+memory T cells and their capacity for secondary expansion.J.Immunol.175:1665–1676.
57.Ciofani,M.,G.C.Knowles,D.L.Wiest,H.von Boehmer,and J.C.Zu
′n ?iga-P?u ¨cker.2006.Stage-speci?c and differential notch dependency at the alphabeta and gammadelta T lineage bifurcation.Immunity 25:105–116.
58.Kang,J.,M.Coles,and D.H.Raulet.1999.Defective development of g /d T cells
in interleukin 7receptor-de?cient mice is due to impaired expression of T cell receptor g genes.J.Exp.Med.190:973–982.
59.Washburn,T.,E.Schweighoffer,T.Gridley,D.Chang,B.J.Fowlkes,D.Cado,
and E.Robey.1997.Notch activity in?uences the alphabeta versus gammadelta T cell lineage decision.Cell 88:833–843.
60.Garbe,A.I.,A.Krueger,F.Gounari,J.C.Zu ′n ?iga-P?u ¨cker,and H.von Boehmer.
2006.Differential synergy of Notch and T cell receptor signaling determines alphabeta versus gammadelta lineage fate.J.Exp.Med.203:1579–1590.
61.Melichar,H.,and J.Kang.2007.Integrated morphogen signal inputs in gam-madelta versus alphabeta T-cell differentiation.Immunol.Rev.215:32–45.62.Hayday,A.C.,and D.J.Pennington.2007.Key factors in the organized chaos of
early T cell development.Nat.Immunol.8:137–144.
63.Hayes,S.M.,and P.E.Love.2007.A retrospective on the requirements for
gammadelta T-cell development.Immunol.Rev.215:8–14.
https://www.doczj.com/doc/794309662.html,uritsen,J.P.,M.C.Haks,J.M.Lefebvre,D.J.Kappes,and D.L.Wiest.2006.
Recent insights into the signals that control alphabeta/gammadelta-lineage fate.Immunol.Rev.209:176–190.
65.Ciofani,M.,and J.C.Zu ′n ?iga-P?u ¨cker.2005.Notch promotes survival of pre-T cells at the b -selection checkpoint by regulating cellular metabolism.Nat.
Immunol.6:881–888.
66.Gangadharan,D.,https://www.doczj.com/doc/794309662.html,mbolez,A.Attinger,Y.Wang-Zhu,B.A.Sullivan,and
H.Cheroutre.2006.Identi?cation of pre-and postselection TCRalphabeta+intraepithelial lymphocyte precursors in the thymus.Immunity 25:631–641.
67.Gallo,E.M.,M.M.Winslow,K.Cante
′-Barrett,A.N.Radermacher,L.Ho,L.McGinnis,B.Iritani,J.R.Neilson,and G.R.Crabtree.2007.Calcineurin sets the bandwidth for discrimination of signals during thymocyte development.Nature 450:731–735.
68.Pennington, D.J., B.Silva-Santos,T.Silberzahn,M.Esco
′rcio-Correia,M.J.Woodward,S.J.Roberts,A.L.Smith,P.J.Dyson,and A.C.Hayday.2006.Early events in the thymus affect the balance of effector and regulatory T cells.Nature 444:1073–1077.
69.Baldwin,T.A.,K.A.Hogquist,and S.C.Jameson.2004.The fourth way?
Harnessing aggressive tendencies in the thymus.J.Immunol.173:6515–6520.70.Zhang,L.,N.Jin,M.Nakayama,R.L.O’Brien,G.S.Eisenbarth,and
W.K.Born.2010.Gamma delta T cell receptors confer autonomous re-sponsiveness to the insulin-peptide B:9-23.J.Autoimmun.34:478–484.
71.Bonneville,M.,R.L.O’Brien,and W.K.Born.2010.Gammadelta T cell ef-fector functions:a blend of innate programming and acquired plasticity.Nat.Rev.Immunol.10:467–478.
The Journal of Immunology
837
by guest on November 27, 2015
https://www.doczj.com/doc/794309662.html,/Downloaded from