Proc. Natl. Acad. Sci. USAVol. 92, pp. 4763-4767, May 1995Immunology

Bclx regulates the survival of double-positive thymocytesAVERIL MA*, JOHN C. PENAt, BRIAN CHANGt, ELISABETH MARGOSIAN*, LAURIE DAVIDSON*,FREDERICK W. ALT**, AND CRAIG B. THOMPSONt*Howard Hughes Medical Institute, Children's Hospital, and Center for Blood Research, Department of Genetics, Harvard Medical School, Boston, MA 02115;

and tHoward Hughes Medical Institute, Committee on Immunology, and the Gwen Knapp Center for Lupus and Immunology Research, Departments ofMedicine and Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637

Contributed by Frederick W Alt, January 26, 1995

ABSTRACT The belx gene has been shown to regulateprogrammed cell death in- vitro. We now show that Bclxexpression increases dramatically when T cells differentiatefrom CD4- CD8- (double negative) thymocytes to CD4+CD8+ [double positive (DP)] thymocytes. In contrast single-positive (SP) thymocytes express negligible amounts of Bclxprotein. This expression pattern contrasts with that of Bcl2,which is present in double-negative thymocytes, down-regulated in DP thymocytes, and reinduced upon maturationto SP thymocytes. Elimination of Bcix by gene targetingdramatically shortens the survival of DP thymocytes but notthe survival of SP thymocytes or peripheral SP T cells. Thesedata suggest that the induction of Bclx during thymic matu-ration plays a critical role in regulating the length of time DPthymocytes survive in the absence of selection.

During T-cell development, immature T cells in the thymusundergo a complex selection process during which a propor-tion of maturing T cells die by programmed cell death (PCD)(1). Thymocytes lacking both CD4 and CD8 surface antigens[CD4- CD8- or double-negative (DN) cells] mature to thy-mocytes bearing both CD4 and CD8 surface antigens [doublepositive (DP) cells]. DP cells represent 80-85% of all thymo-cytes and are sensitive to PCD when exposed to glucocorti-coids, radiation, or cross-linking of T-cell receptor/CD3 mol-ecules (2-5). Most DP cells die via PCD in vivo, either due tonegative selection or to neglect, the lack of positive selection(6-8). A minority ofDP thymocytes undergo positive selectionand mature to the single-positive (SP) stage, where theyexpress either CD4 or CD8 but not both.The protooncogene bc12 was the first gene studied in the

context of PCD regulation during lymphoid development. Bc12was originally cloned at the chromosomal breakpoint oft(14;18)-bearing B-cell lymphomas (9, 10). Bcl2 protects some,but not all, factor-dependent cell lines from PCD in vitro, andderegulated bc12 expression prolongs the survival of B cells intransgenic mice (11-13). In the thymus, Bcl2 expression ispredominantly restricted to mature SP thymocytes found in themedulla and is largely absent from DP thymocytes that residein the cortex. Bcl2 expression is up-regulated in DP thymocytesafter positive selection and may assist them in differentiatingto SP cells (14).Forced expression of bc12 in DP thymocytes of transgenic

mice enhances the resistance of these cells to 'y-radiation andglucocorticoid-induced PCD but does not prevent the physi-ological elimination of DP thymocytes by negative selection(15, 16). Elimination of bc12 by gene targeting results inprogressive apoptosis of B and T lymphocytes beginning at 3to 4 weeks of age (17, 18). In addition, bc12-- SP thymocytesand mature peripheral T lymphocytes demonstrate increasedsensitivity to PCD by -y-irradiation and dexamethasone. How-ever, T-cell development occurs normally in these animals, and

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bc12-/- DP thymocytes display no increased sensitivity to PCDin the absence of PCD-inducing agents (17, 18). These trans-genic experiments indicate that Bcl2 regulates the survival oflymphocytes in response to a variety of stimuli that inducePCD. However, these experiments also indicate that lympho-cyte survival is not entirely dependent upon Bc12 and thatadditional genes may be important in regulating PCD duringlymphocyte development.The bclx gene was cloned by screening cDNA from avian

lymphoid cells for genes homologous to murine bc12 (19). Twoforms of bclx mRNA have been observed. The longer formencodes a larger protein, Bclxl, which protects factor depen-dent cells against PCD, whereas a shorter protein, Bc1x5,counters the protective effect of Bcl2 in similar cells. Bclx1shares greatest homology with Bcl2 within an amino aciddomain that is deleted from BclxS as a result of alternativesplicing (19). To determine whether Bclx proteins play a rolein lymphocyte development, we studied Bclx protein expres-sion at various stages of T-cell differentiation. We have alsomutated the bclx gene in embryonic stem (ES) cells by genetargeting and studied the effects of bclx deficiency on lym-phocyte development.

MATERIALS AND METHODSGeneration ofAnti-Bclx Monoclonal Antibodies and Immu-

noanalyses of Lymphocytes. Recombinant Bclx proteins wereproduced in Escherichia coli and used to generate murinemonoclonal antibodies. Several monoclonal antibodies wereidentified that reacted with murine Bclxl and BcIxS, in additionto human Bclx proteins. For immunoblotting, single-cell sus-pensions of lymphocytes from lymph nodes (LN) or thymusesof 6-week-old CD1 mice were lysed in 1% Nonidet P-40/1%deoxycholate/0.1% SDS with protease inhibitors. The proteinconcentration was determined by bicinchoninic acid assay(BCA, Pierce), and 25 ,ug of protein per sample was separatedin SDS/15% polyacrylamide gels. Proteins were blotted ontonitrocellulose and probed with monoclonal antibodies directedagainst either Bclx (2A1) or Bc12 (3F11, PharMingen) proteins.Reactive proteins were detected by enhanced chemilumines-cence (ECL, Amersham).Gene Targeting of bclx. To target the bclx gene in ES cells,

a murine clone of the bclx gene was isolated from a 129SVgenomic library (Stratagene). Targeting constructs (20) wereengineered by using this bclx clone, a pGK-thymidine kinase(TK) expression casette and either a pGK-neomycin-TK ex-pression casette or a pGK-puromycin-TK expression casette(provided by P. Laird and R. Jaenisch, Whitehead Institute,Cambridge, MA) to permit serial targeting both bclx alleles. JlES cells transfected with a pGK-neomycin-bearing targetingconstruct (see below) were selected in G418 at 0.4 mg/ml and

Abbreviations: PCD, programmed cell death; DN, double negative;DP, double positive; SP, single positive; LN, lymph node(s); ES cell,embryonic stem cell.*To whom reprint requests should be addressed.

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2 mM 2'fluoro-5-iodo-arabinofuranosyluracil (FIAU, pro-vided by R. Jaenisch), and genomic DNA from resistant cloneswas screened by Southern analysis for homologous integrantsin one allele. The second bclx allele of two independent cloneswas mutated in a similar fashion by transfection with apGK-puromycin-based construct, followed by selection in thepresence of puromycin at 2 ,tg/ml and 2 mM FIAU ony-irradiated neomycin and puromycin-resistant STO cells. EScells from two independently targeted bclx+l- clones and twoindependent bxr-/- clones were injected into recombinaseactivating gene (Rag2-/-) blastocysts, blastocysts were trans-ferred to foster mothers, and chimeric offspring were identi-fied for further analysis as described (21, 22). bclx+l- chimera(seven analyzed) contained 15-65 x 106 thymocytes, 10-20 x106 splenic T cells, 3.0-8.0 x 106 LN T cells, and 2.5-3.5 x 106LN B cells, and 25-36 x 106 splenic B cells per mouse; whereasbclx-l- chimera (six analyzed) contained 10-45 x 106 thy-mocytes, 8-15 x 106 splenic T cells, 2.0-6.0 x 106 LN T cells,0.1-0.2 x 106 LN B cells, and 0.4-1.8 x 106 splenic B cells permouse.Lymphocyte Survival Studies. Thymocytes or LN T cells

were diluted to 1 X 106 cells per ml in RPMI 1640 medium/15% fetal calf serum/50 AM 2-mercaptoethanol and culturedin 96-well U-bottom tissue culture plates. Irradiation was doneby using an Astrophysics (Harbor City, CA) 120D y-irradiationsource. Anti-CD3 antibody treatment was done by precoating96-well plates with 2C11 anti-CD3s monoclonal antibody at 10jig/ml (23) for 1 hr at 37°C.

Cell Cytometry Analyses and Sorting. CD4 and CD8 anti-gens were detected by staining lymphocytes with fluoresceinisothiocyanate (FITC) conjugated RM4-5 (anti-CD4) and phy-coerythrin conjugated 53-6.7 (anti-CD8) monoclonal antibodiesat 10 ,ug/ml final concentration (PharMingen) for 30 min on icewashing cells, and analyzing them on a Becton Dickinson FAC-Scan. Cell cytometry sorting of stained cells was done by using aBecton Dickinson FACStar Plus.

RESULTSExpression of Bclx Proteins in Vivo. Analysis of bclx expres-

sion in chicken tissues indicated that Bclx mRNA was ex-pressed at highest levels in the thymus (19). To examine Bclxprotein expression during murine T-cell maturation, we per-formed immunoblot analyses of lysates from thymocytes andsorted SP LN T cells. Bclx protein was readily detected inthymocytes but was not detected in mature LN T cells, whereasBc12 protein was preferentially expressed in mature T cells

A B X C

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probe A

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FIG. 2. Strategy for homozygous deletion of Bclx in ES cells. (A)Endogenous bclx locus (top), neomycin-based (KOlneo construct,middle), and puromycin-based (KOlpuro construct, bottom) targetingvectors (boldface lines).A 1.6-kbBamHI-Sac II flanking probeA usedfor screening for homologous integration is indicated below. Predictedsizes of Sac I/Cla I-digested DNA hybridizing to probe A: endogenouslocus, 8.7 kb; neo mutant, 4.7 kb; puro mutant, 3.3 kb. S, Sac I; C, ClaI; B, BamHI; X, Xba I. (B) Sac I/Cla I-digested DNA from ES clonesbearing single (bcix+-) or double (bclx/-) mutations at the bcixlocus, hybridized to probe A. puro, Puromycin; neo, neomycin.

(Fig. 1). The Bclxl form of the protein was predominant inthymocytes (Fig. 1A).To determine which thymocytes expressed Bclx, thymocytes

from 6-week-old CD1 mice were sorted by cell cytometry intoSP and DP populations. Thymocytes from 8-week-old Rag2-/-mice-whose T cells do not mature beyond the DN stage ofdevelopment-were harvested to obtain a pure source of DNthymocytes (21). DN Rag2-/- thymocytes were stained withthe 7D4 anti-interleukin 2 receptor a chain monoclonal anti-

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FIG. 1. Expression of Bclx and Bc12 proteins in T-cell subsets. (A) Bclx and Bc12 expression in immature vs. mature T cells. Twenty-fivemicrograms of lysate from sorted SP LN T cells or thymocytes, or FL5.1.2 cells cotransfected with bclxl and bclx, expression constructs (20) assayedby immunoanalysis for Bclx and Bcl2 expression. (B and C) Expression of Bclx and Bc12 proteins in thymocyte subsets. Twenty-five microgramsof lysate from Rag2-/- DN thymocytes, sorted SP and DP thymocytes from CD1 mice, and control FL5.1.2 cells transfected with both bclxl andbclxs expression constructs (20) were assayed for Bclx and Bcl2 expression as in A.

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body and found to be >95% positive by cell cytometry analysis,indicating that these cells were mostly T lymphocytes and notstromal cells (data not shown). Probing duplicate immunoblotsof sorted thymocytes with 2A1 or 3F11 monoclonal antibodiesdemonstrated that Bclx expression in the thymus is inverselycorrelated with Bcl2 expression: Bclx is expressed preferen-tially in DP thymocytes (compared with DN or SP thymo-cytes), whereas Bcl2 is expressed preferentially in DN and SPthymocytes (compared with DP thymocytes) (Fig. 1 b and c).These data suggest that Bclx is induced as cells differentiateinto DP cells and down-regulated upon maturation to SP cells.

Elimination of Bclx by Gene Targeting. We disrupted bothcopies of the bclx gene in Jl ES cells by successively targetingthe two alleles with vectors in which the first exon of bclx wasreplaced with either a pGKneo (KOlneo) or a pGKpuro(KOlpuro)-based targeting construct (Fig. 2 a and b). Ho-mologous targeting events occurred at a frequency of -1:20with both the first (KOlneo) and second (KOlpuro) con-structs. The isolation of bclx-- ES cells demonstrated thatbclx, which is expressed in normal ES cells (data not shown),is not essential for the survival of this undifferentiated celltype.Lymphocyte Development from bcl-- ES Cells. We tested

the ability of bclx-l- ES cells to differentiate into lymphocytesby the Rag2-/- blastocyst complementation method, whichentails generating chimeric mice by injection of Rag2-/-blastocysts with normal or mutant ES cells (22). In such

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chimera, all T cells beyond DN stage and all B cells beyond thepro-B cell stage must derive from the injected ES cells (22). Weidentified seven chimeric mice from two independent bclx+l-ES cell clones and six chimeric mice from two independentbclx-- ES cells. Both bcLb+/- and bclxb-- chimera containedsubstantial numbers of thymocytes and peripheral T cells,indicating that bclx is not essential for T-cell development(Figs. 3 and 4; see Materials and Methods). However, althoughbcLb+/- chimera had substantial peripheral B-cell numbers,bcl-- chimera contained only very low numbers of periph-eral IgM+ B cells (see Materials and Methods), the existence ofwhich was also confirmed in two analyzed bcl-x- chimera bylow, but detectable, levels of serum IgM levels (2.5-5.7 ,g/ml;data not shown). These data demonstrate that Bclx is notrequired for development of mature T and (probably) B cells.

Survival of bcl-- Thymocytes. To assay survival, thymo-cytes were placed in culture, and the live cells were counted (bytrypan blue exclusion) at serial time points. With increasedtime in culture, a marked survival advantage of bcLx+l- overbcl-- thymocytes became apparent (Fig. 3A). Treatment ofthymocytes with dexamethasone, y-irradiation, or anti-CD3santibody is known to induce PCD of DP thymocytes. Thus,bclx+/- and bclx-/- thymocytes were cultured on anti-CD3e-coated wells (Fig. 3B), cultured with 100 nM dexamethasone(Fig. 3C), or treated with 500 rads of y-irradiation (1 rad = 0.01Gy) before culture in medium alone (Fig. 3D). In all condi-tions, bclx-l- thymocytes died more rapidly than bclx+/- cells.

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FIG. 3. Sensitivity of bclx-l- thymocytes to spontaneous cell death. Serial cell counts were performed from thymocyte cultures from 3-week-oldbclx+l- (o) and bclx-l- (m) chimeric mice. The percentage viability of thymocytes treated with media alone (A), anti-CD3e monoclonal antibody(B), 100 nM dexamethasone (C), or 500 rads of -y-irradiation (D) is expressed as a percentage of initial cell number. Data represents the meanof three pairs of age-matched bclx-/- and bclx+/--derived chimera. (E) Serial cell cytometry analyses of phycoerythrin-anti-CD4- andFITC-anti-CD8-stained thymocyte cultures from bclx-/- or bclx+/- chimeras. Percentage of gated cells in DP populations is indicated on eachscan. Cell cytometry analyses were done after 0, 3, 6, 20, and 40 hr of culture.

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FIG. 4. Lack of sensitivity of bclx-l- SP T cells to cell death. Serial cell counts were done from LN cell cultures from 3-week-old bclx-/ ()and bclx+l- (o) chimeric mice. The percentage viability of LN T cells treated with media alone (A), 10 nM dexamethasone (B), or 500 rads ofy-irradiation (C) is expressed as a percentage of initial cell number. Data represents mean values from four pairs of age-matched bclx-/- orbclx+/--derived chimera from two independent clones. (D) Representative cell cytometry analyses of bclx-l- and bclx+l- LN T cells stained withphycoerythrin-anti-CD4 and FITC-anti-CD8 monoclonal antibodies. Percentage of gated cells in CD4+ and CD8+ populations are indicated oneach scan. Cell cytometry analyses were done at 0, 3, and 20 hr of culture.

These data suggest that Bclx is involved in regulating thymo-cyte survival. To determine whether differences in survivalcorrelate with the expression pattern of Bclx, we stained cellsat various time points after in vitro culture to distinguish SPfrom DP T cells; these analyses revealed that bclx-l- DPthymocytes survive less well than bclx+/- DP cells, whereasbclx-l- SP thymocytes survived as well as bclx+l- SP thymo-cytes. Thus, Bclx selectively regulates the survival of DPthymocytes (Fig. 3E).

Survival of Peripheral bclx'-- T Cells. Peripheral T cellsexpress very little Bclx protein (Fig. 1). To determine the con-sequences of Bclx deficiency upon peripheral T-cell survival, LNcells from bclxr+- and bcb-l- chimera were placed directly intoculture, exposed to 500 rads of y-irradiation before culture orplaced into cultures containing 10 nM dexamethasone. Survivalof total bclx+l- SP T cells was comparable to bclx-/- SP T cellsin all conditions (Fig. 4A-C), and no relative survival advantagewas observed for CD4+ or CD8+ bclx+l- or bclx-/- cells (Fig.4D). Thus, Bclx is not necessary for protecting resting SP T cellsfrom spontaneous or induced PCD.

DISCUSSIONWe have studied the role of Bclx during thymocyte differen-tiation by characterizing its expression pattern in vivo and bystudying the consequences of its loss of function in thymocytes

by gene targeting. Our gene-targeting strategy deletes theentire first exon of bclx and should thus eliminate both BclxSand Bclxl proteins, as well as the product of an unspliced formof Bclx, BclxB, which is also expressed in the thymus (24). Wehave found that Bclxl is the predominant form expressed inimmature T cells. Thus it is probable that Bclxl, which protectscells against PCD, is the major Bclx protein regulating cellsurvival in T-cell development. In confirmation of this notion,loss of bclx selectively compromises the survival of DP thy-mocytes.The structural and functional homology of Bclxl with Bc12

suggested that Bclxl, like Bc12, might protect immature T cellsfrom PCD. We have found multiple differences in the expres-sion of these two proteins: Bc12 is expressed in DN and SPthymocytes but is expressed only in rare DP thymocytes (25,26; Fig. 1), whereas Bclxl is almost exclusively expressed in DPthymocytes. This divergent expression pattern suggests thatthese two proteins have evolved distinct roles in T-cell devel-opment. Although both proteins allow factor-dependent celllines to resist PCD in vitro, differential use of Bcl2 vs. Bclxl mayallow T cells at different stages of development to resist differentdeath inducing stimuli in vivo.An emerging picture of Bcl2 and Bclx expression and

function in T-cell development would suggest that DN thymo-cytes down-regulate Bc12 expression and induce Bclx expres-sion upon differentiating to DP thymocytes. These DP cells are

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sensitive to PCD induction via negative selection because oftheir relative deficiency of Bcl2, and their spontaneous survivaldepends on the maintenance of Bclxl expression. The majorityof these cells ultimately die by neglect, due either to a reductionof Bclxl expression or another factor compromising Bclx1 function.Positive selection occurs in a minority of these cells, which leadsto the induction of Bcl2 and down-regulation of Bclx expression.

In contrast to its role in immature T cells, loss of Bclx doesnot affect the survival of mature SP T cells. This finding isconsistent with the negligible expression of Bclx in sorted Tcells from normal mice. By contrast, mature bc12-- T lym-phocytes are more susceptible to PCD induction. Thus, al-though Bclx regulates the lifespan of DP thymocytes beforeselection, Bc12 is more important for maintaining survival ofpositively selected SP T cells in the thymus and in restingperipheral T cells.The Rag2-/- blastocyst complementation system allowed us

to study the role of Bclx proteins in lymphoid developmentindependent of their role in the development of other tissues.Loss of Bclx clearly did not prevent the appearance of matureT cells. Thus, despite their complementary expression pat-terns, neither Bc12 nor Bclx is absolutely required for matureT-cell development. We have detected only very low numbersof peripheral B cells in bclx-/- chimera. As immature B220+,IgM- B cells express Bclxl protein at high levels (data notshown), it is possible that the poor reconstitution of the B-celllineage observed in bclx-l- chimera may be due to an adverseeffect of Bclx deficiency upon the survival of immature B cells.However, prior experience has suggested that factors otherthan a specifically introduced mutation can lead to preferentialreconstitution ofT versus B cells from a given ES cell clone inthe context ofRag2-deficient blastocyst complementation (22).

We thank Wojciech Swat and Peter Laird for helpful discussions,advice, and gifts of purified interleukin 2, anti-CD3e antibody, andpGKpuromycin vector. We thank Julie Auger for assistance in cellsorting. We thank Harvey Cantor and Stan Korsmeyer for criticallyreading the manuscript. This work was supported by the HowardHughes Medical Institute (F.W.A. and C.B.T.) and National Institutesof Health Grants CA-42335 and AI-20047 (F.W.A.) and AI-35294(C.B.T.). A.M. and E.M. are supported by a James S. McDonnellFoundation Award (A.M.). J.C.P. was supported by National Institutesof Health Research Supplement for Underrepresented MinoritiesP01AI35294. B.C. was supported by National Institutes of HealthMedical Scientist Training Program Training Grant T32GM07281.

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