Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors

transcript

Deregulated expression of RasGRP1 initiates thymic lymphomagenesis

independently of T-cell receptors

Mark B Klinger1,2,3, Benoit Guilbault1,2,4, Rebecca E Goulding1,2 and Robert J Kay*,1,2

1Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada; 2Department of Medical Genetics, University ofBritish Columbia, Vancouver, BC, Canada

RasGRP1 is a Ras-specific exchange factor, which isactivated by T-cell receptor (TCR) and promotes TCR-dependent positive selection of thymocytes. RasGRP1 ishighly expressed on most T lymphocytic leukemias and isa common site of proviral insertion in retrovirus-inducedmurine T-cell lymphomas. We used RasGRP1 transgenicmice to determine if deregulated expression of RasGRP1has a causative role in the development of T-cellmalignancies. Thymic lymphomas occurred in threedifferent RasGRP1 transgenic mouse lines. Thymocytetransformation correlated with high transgene expressionin early stage lymphomas, indicating that deregulatedRasGRP1 expression contributed to the initiation oflymphomagenesis. Expression of the positively selectableH-Y TCR accelerated lymphomagenesis in RasGRP1transgenic mice. However, the transformed thymocyteslacked markers of positive selection and lymphomasoccurred when positive selection was precluded bynegative selection of the H-Y TCR. Therefore, initiationof lymphomagenesis via RasGRP1 was not associatedwith TCR-dependent positive selection of thymocytes.Thymic lymphomas occurred in RasGRP1 transgenic/Rag2�/� mice, demonstrating that neither TCR norpre-TCR were required for RasGRP1-driven lymphoma-genesis. The RasGRP1 transgene conferred pre-TCR-independent survival and proliferation of immature thymo-cytes, suggesting that deregulated expression of RasGRP1promotes lymphomagenesis by expanding the pool ofthymocytes which are susceptible to transformation.Oncogene (2005) 24, 2695–2704. doi:10.1038/sj.onc.1208334Published online 27 December 2004

Keywords: T lymphocytes; T-cell receptors; leukemia;lymphoma

Introduction

Thymocytes pass through a series of developmentalprogressions which ensure the production of a large andgenetically diverse population of immunologically com-petent T cells. The expansion, genetic diversification andimmunological selection of thymocytes has to berigorously regulated to prevent the emergence of eitherself-reactive or malignant clones. At the b selectioncheckpoint, double negative (DN, CD4� CD8�) thymo-cytes are challenged for T-cell receptor (TCR)b func-tionality. If TCRb is productively rearranged, itassembles with CD3 and pTa to form a constitutivelysignaling pre-TCR which confers cell division, apoptosissuppression and differentiation into the intermediatesingle positive (ISP, CD4þ or CD8þ ) and then doublepositive (DP, CD4þCD8þ ) stages of thymocyte devel-opment (Kruisbeek et al., 2000; Michie and Zuniga-Pflucker, 2002). Subsequent rearangement and surfaceexpression of TCRa generates a diversity of TCRs. DPthymocytes bearing self-reactive TCRs are eliminated bynegative selection, while those with TCRs capable ofinteracting with MHC in the absence of antigen undergopositive selection, resulting in survival, limited prolifera-tion and maturation into single positive (SP, CD4þ orCD8þ ) thymocytes (Sebzda et al., 1999; Hogquist,2001).

RasGRP1 is a Ras GTPase-specific guanine nucleo-tide exchange factor which is positively regulated byTCR at both the transcriptional and biochemical levels.Pre-TCR ligation or TCR–MHC interactions result intranscriptional induction of the RasGRP1 gene (Nor-ment et al., 2003; Tiong Ong et al., 2003), andbiochemical activation of RasGRP1 is dependent onsignal transduction from TCR via phospholipase Cg1(Ebinu et al., 2000; Bivona et al., 2003). AlthoughRasGRP1 is not required for pre-TCR-directed bselection in normal mice (Dower et al., 2000), constitu-tive transcription of RasGRP1 from a transgene conferspartial developmental progression up to the DP stage inthe absence of pre-TCR (Norment et al., 2003). Theefficiency of TCR-directed positive selection of DPthymocytes is severely compromised in RasGRP1-deficient mice (Dower et al., 2000; Priatel et al., 2002;Layer et al., 2003). Conversely, RasGRP1 transgenicmice have increased numbers of CD8 SP thymocytesthat have enhanced proliferative and survival responses

Received 11 May 2004; revised 20 September 2004; accepted 28 October2004; published online 27 December 2004

*Correspondence: R Kay, Terry Fox Laboratory, British ColumbiaCancer Agency, 600 West 10th Avenue, Vancouver, BC, Canada V5Z4E6; E-mail: rkay@bccrc.ca3Current address: Department of Microbiology and Immunology,University of California, San Francisco, 513 Parnassus Avenue, SanFrancisco, CA 94143-0414, USA4Current address: StemCell Technologies Inc., 570 West 7th Avenue,Vancouver, BC, Canada V5Z 1B3

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to TCR ligation (Norment et al., 2003). Thus, RasGRP1serves to transduce signals from pre-TCR and TCRwhich allow thymocytes to pass through the two criticalcheckpoints during T-cell development, b selection andpositive selection.

Activating point mutations in N-Ras occur in about10% of human T lymphocytic leukemias (Ahuja et al.,1990; Yokota et al., 1998; Kawamura et al., 1999), andtransgenic mice expressing mutationally activated N-Ras or H-Ras have high incidences of thymic lympho-mas (Dunbar et al., 1991; Hawley et al., 1995; Swat et al.,1996; Curtis et al., 1997; Adams et al., 1999). RasGRP1is one of many Ras-regulating proteins (Quilliam et al.,2002) whose structural mutation or aberrant expressioncould potentially mimic the oncogenicity of Ras muta-tions. RasGRP1 is highly expressed in most cases ofhuman acute T lymphocytic leukemias (Yeoh et al.,2002), which originate predominantly from transformedthymocytes (Asnafi et al., 2002). More direct evidenceindicating that RasGRP1 has the potential to act as adominant oncogene in T cells has been obtained fromanalyses of murine lymphomas which arose as aconsequence of retroviral insertional mutagenesis (Liet al., 1999; Kim et al., 2003). Of 48 T-cell lymphomasoccurring after infection of neonatal mice with the SL3retrovirus, five had proviral insertions near theRasGRP1 gene (Kim et al., 2003). Although theselymphomas have not been characterized in terms of cellphenotypes or RasGRP1 expression, it is expected thatthey arose due to the imposition of the proviral patternof expression on the adjacent RasGRP1 gene.

In this paper, we demonstrate that deregulatedexpression of RasGRP1 via a transgene initiates thedevelopment of thymic lymphomas and T-cell leuke-mias. By crossing the RasGRP1 transgene onto geneticbackgrounds providing expression or absence of posi-tively selectable TCRs, we found that lymphomagenesisvia RasGRP1 was not dependent on positive selection,appeared to originate in immature DN or ISP thymo-cytes, and occurred independently of either TCR or pre-TCR. These results indicate that the oncogenic potentialof RasGRP1 during thymocyte development reflects itsability to confer survival and proliferation on immaturethymocytes, rather than its enhancement of TCR-directed positive selection of DP and SP thymocytes.

Results

RasGRP1 transgenic mice develop thymic lymphomas

Three different RasGRP1 transgenic mouse lines,AM1268, 13H and 293H, were analysed for thedevelopment of T-cell malignancies. There was anelevated death rate in all three lines, most notably inthe 13H line where 50% of the mice died within 130 daysof birth (Figure 1). Death was typically preceded by aperiod of 1–2 days during which the mouse would begenerally healthy but would show signs of laboredbreathing. Mice with these symptoms were killed andanalysed. In all three lines, the afflicted mice invariably

had thymic lymphomas, with elevated numbers ofthymocytes ranging from two- to 10-fold above thosein nontransgenic littermates. The phenotypes of arepresentative set of six thymic lymphomas from the13H line are shown in Figure 2a, along with anontransgenic thymus and a thymus from a healthy13H transgenic mouse. The lymphomas had variablephenotypes, with expansion occurring in combinationsof DN, DP and/or SP thymocytes. The lymphomascontained a high proportion of proliferative cells, whilenormal (nonlymphomic) transgenic thymocytes were

Figure 1 Lymphoma incidence in RasGRP1 transgenic lines. Thefigure shows the survival of groups of 16 RasGRP1 transgenic miceof the 13H or 293H lines, 11 RasGRP1 transgenic mice of theAM1268 line and 16 nontransgenic littermates of the 13H line.Circles (13H), triangles (293H) and diamonds (AM1268) indicatedeaths of individual mice, or the killing of a mouse with a largelymphoma. Both deceased AM1268 mice had large thymiclymphomas

Figure 2 Phenotypes of thymic lymphomas in RasGRP1 trans-genic mice. (a) CD4�CD8 flow cytometry profiles of totalthymocytes are shown. The age of the mouse in days is indicatedin the box at lower left, and the extent of thymus enlargementrelative to nontransgenic littermate controls is indicated in the boxat lower right. The top left profile is a nontransgenic mouse of the13H line, and the top right profile is a normal 13H RasGRP1transgenic mouse with no detectable thymocyte transformation.The other six profiles show 13H RasGRP1 transgenic mice whichhad thymic lymphomas. Fluorescence intensities of CD4 and CD8staining are on log scale with unit increments. (b) Thymocytes froma normal RasGRP1 transgenic mouse of the 13H line (mouse A in(a)) as well as lymphoma-bearing 13H mouse (mouse G in (a)) andits nontransgenic littermate were permeabilized and stained withpropidium iodide, and then analysed by flow cytometry to measureDNA content. The percentages of cells with greater than 2N DNAcontent (in S, G2 or Mphases of the cell cycle) or less than 2 NDNA content (apoptotic) are indicated. Propidium iodide fluores-cence intensity is on a linear scale with unit increments. The verticalaxis on this and all other histograms indicates cell number on alinear scale. (c) Histograms of forward light scatter for theindicated thymocyte subsets of mouse G (dark line) and itsnontransgenic littermate (shaded). Forward light scatter intensity ison a linear scale with unit increments. (d) Expression of CD25,CD62L, CD69 and CD5 on DP thymocytes of mouse G (dark line)and its nontransgenic littermate (shaded). Fluorescence intensity ofantibody staining are on log scales with unit increments. (e)CD4�CD8 profiles of the spleens of mouse G and its nontrans-genic littermate, showing percentages of CD4þCD8þ cells

Thymic lymphomagenesis via RasGRP1MB Klinger et al

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indistinguishable from nontransgenic thymocytes intheir proportions of proliferative and apoptotic cells(Figure 2b). Transformed cells within lymphomas werealso distinguished from normal thymocytes by their

enlargement as detected by high forward light scatter(Figure 2c). High light scatter was particularly effectivefor detecting transformation of DP thymocytes, asthymocytes at this developmental stage are normallyalmost all small and nonproliferative.

Thymocytes normally lose expression of CD25 priorto the DP stage (Michie and Zuniga-Pflucker, 2002).However, there was high CD25 expression on DPthymocytes in seven out of nine 13H lymphomas(example in Figure 2d). Induction of CD25 expressionaccompanies T-cell activation via TCR. However,another marker of T-cell activation, loss of CD62Lexpression (Dailey, 1998), did not accompany thymo-cyte transformation in RasGRP1 transgenic thymocytes(Figure 2d). Activation of DP thymocytes via positivelyselecting TCR triggers increased expression of CD69and CD5 (Azzam et al., 1998; Anderson et al., 1999), butboth of these markers were low in transformed DPthymocytes (Figure 2d). Therefore, while the trans-formed DP thymocytes are activated in terms of beingproliferative and enlarged, they do not express a full setof markers associated with TCR-dependent activation.

In some mice with large thymic lymphomas, thespleen, lymph nodes and/or bone marrow were infil-trated with enlarged lymphocytes resembling the trans-formed cells in the thymic lymphoma (Figure 2e, anddata not shown). Cells from thymic lymphomas orinfilitrated lymph nodes survived and proliferated inculture medium, resulting in the establishment after 2–3weeks of rapidly dividing, clonable cell lines with CD4and CD8 expression patterns resembling those of thetransformed cells which initiated the cultures (data notshown). Therefore, the hallmarks of transformation ofthe thymic lymphoma cells included loss or distortion ofnormal differentiation, deregulated proliferation, dis-semination as peripheral leukemias, and ability tosurvive and proliferate indefinitely in culture in theabsence of supportive cytokines or stromal cells.

We used flow cytometric analysis of TCR Vb usage(Cleverley et al., 2000) to determine the clonality oflymphomas arising in RasGRP1 transgenic mice.Normally, about 15% of SP thymocytes in C57BL/6mice have the Vb8 rearrangement of the TCRb gene. If apopulation of cells is clonally derived from a singlethymocyte which had undergone TCRb rearrangementand allelic exclusion to fix its Vb usage, the populationwill exhibit loss of heterogeneity of Vb expression. Inabout 85% of cases, this will result in the absence of Vb8expression in the clonal population, and in about 15%of cases all of the SP cells will express Vb8. Fourlymphomas from 13H transgenic mice contained trans-formed SP thymocytes with moderate to high TCRbexpression and therefore could be assessed for clonality.In all four cases, the CD8 SP thymocytes of thelymphomas had ratios of TCR-Vb8 to total TCRb thatwere 10–50-fold lower than the ratio of 0.15 observedfor normal CD8 SP thymocytes (Table 1). The lack ofrepresentation of Vb8 indicates that the lymphomasoriginated from one or a few thymocytes which hadundergone TCRb rearrangement and had subsequentlyundergone clonal expansion.

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High transgenic RasGRP1 expression correlates with theinitiation of lymphomagenesis

The transgene-encoded RasGRP1 protein is expressedat moderate to low levels in all thymocyte subsets ofnormal (lymphoma-free) mice of the AM1268 line(Figure 3), resulting in a net doubling of RasGRP1protein levels relative to nontransgenic thymocytes(Norment et al., 2003). Transgenic RasGRP1 expressionwas similar in the 293H line except for minimalexpression in DP thymocytes (Figure 3, mouse S),whereas in the 13H line expression of transgenicRasGRP1 protein was detected only in a minor portion

Table 1 Clonality analysis of CD8 SP thymocytes by Vb8 usage

Mouse % TCR-Vb8hi % TCR-bhi Vb8hi/bhi

Aa 13 87 0.15C 0.6 45 0.013D 0.4 24 0.017F 0.2 71 0.003G 0.1 19 0.005

aCD4/CD8 profiles of the thymuses of each mouse are shown in Figure2. Mouse A had a normal thymus with no evidence of lymphomadevelopment, while mice C, D, F and G had lymphomas involvingCD8 SP thymocytes as indicated by increased forward light scatter(Figure 2, and data not shown)

Figure 3 Elevated RasGRP1 expression in transformed thymocytes. CD4�CD8 profiles of RasGRP1 transgenic mice are shown onthe left, with the particular transgenic line indicated. Mice E (shown in Figure 2a) and T had large thymic lymphomas while thethymuses of the other mice were of normal size. On the right are flow cytometric measurements of the HA epitope-tagged transgenicRasGRP1 protein in each thymocyte subset of these mice. Shaded histograms are nontransgenic littermates (CD4�CD8 profiles notshown) and open histograms are the RasGRP1 transgenic mice, with the gates that define the thymocyte subsets of the transgenicsshown on the CD4�CD8 profiles. Fluorescence intensities of anti-HA intracellular staining are on log scales with unit increments. Thehistogram at the bottom shows forward light scatter (linear scale) of DP thymocytes of mouse H, comparing HAhigh (heavy line) toHAlow (shaded)

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of DP thymocytes in normal mice (Figure 3, mouse A).In contrast, transgenic RasGRP1 expression was high inall transformed thymocytes within lymphomas arising inRasGRP1 transgenic mice (e.g. Figure 3, mice E and T).Therefore, lymphomagenesis is accompanied by atransition from low or moderate to high levels ofexpression of the RasGRP1 transgene. The variableincidences of lymphomas in the three different trans-genic lines may reflect an integration site-dependentvariation in the probability of the transgene undergoinga random transition to high expression.

To determine if acquisition of high RasGRP1expression was an initial or late event during lymphomadevelopment, we examined transgenic RasGRP1 expres-sion in transformed versus nontransformed thymocytesin mice with very early stage lymphomas. An example ismouse H of the 13H line, which had a normalcomplement of DN and CD4 SP thymocytes but had asmall increase in DP thymocytes and an atypicalpopulation of CD8þCD4� thymocytes (Figure 3). Mostof the CD8þCD4� thymocytes and a minority of the DPthymocytes in mouse H had high forward light scatter,indicating transformation. In this mouse, all DN andCD4 SP thymocytes had undetectable transgenicRasGRP1 expression, while the atypical CD8þCD4�

thymocytes and a discrete minority of DP thymocyteshad high expression (Figure 3). The DP thymocytes withno detectable expression of transgenic RasGRP1 hadthe low light scatter which characterizes quiescent DPthymocytes (Figure 3, bottom histogram). In contrast,the DP thymocytes with high transgenic RasGRP1expression had high light scatter, indicating transforma-tion. This analysis indicates that the thymus of mouse Hcontained transformed DP and CD8þCD4� thymocyteswith high transgene expression, coexisting with a largernumber of normal DN, DP and CD4 SP thymocyteswhich retained low or nil transgene expression. Theprecise correlation between high RasGRP1 expressionand thymocyte transformation observed in this and allother analysed mice, including other early stagelymphomas, demonstrates that high RasGRP1 expres-sion is an early and thus potentially causative event inlymphomagenesis.

Testing the role of TCR-mediated positive selection inlymphomagenesis via RasGRP1

RasGRP1 is activated in response to TCR ligation(Ebinu et al., 2000; Bivona et al., 2003), and transducessignals that enable TCR-mediated positive selection atthe DP stage (Dower et al., 2000; Priatel et al., 2002;Layer et al., 2003) as well as enhanced survival andTCR-induced proliferation at the SP stage (Normentet al., 2003). This raised the question of whether thelymphomas in RasGRP1 transgenic mice were causedby amplified signaling downstream of TCR, leading toan exaggeration of the survival and proliferativeresponses which confer positive selection. If enhance-ment of positive selection by RasGRP1 plays aninitiating role in lymphoma development, then increas-ing the proportion of thymocytes undergoing positive

selection in RasGRP1 transgenic mice should increasethe incidence of lymphomas. In female mice transgenicfor the MHC class I-specific H-Y TCR, a highproportion of DP thymocytes are positively selectedon H-2Db and differentiate into mature CD8 SPthymocytes (Kisielow et al., 1988). The efficiency ofpositive selection of DP thymocytes expressing the H-YTCR on H-2Db is attenuated in RasGRP1-deficient mice(Priatel et al., 2002) and is enhanced in AM1268RasGRP1 transgenic mice (data not shown). Expressionof the H-Y TCR markedly increased and accelerated theincidence of thymic lymphomas in female mice of theAM1268 RasGRP1 transgenic line (Figure 4, incomparison to Figure 1). The phenotype of oneRasGRP1 Tgþ /H-Y TCRþ female with a large thymiclymphoma is shown in Figure 5. The lymphoma cells area combination of DP and CD8 SP, as might be expectedif they arose from exaggerated positive selection of theH-Y TCR which directs survival of DP thymocytes andtheir differentiation into the CD8 lineage. However,none of the CD8 SP cells of this mouse expressed CD69,which is induced in response to RasGRP1-dependentpositive selection of the H-Y TCR (Priatel et al., 2002).The lymphoma CD8 SP cells also differed from thepositively selected CD8 SP thymocytes of H-Y TCRtransgenic mice by being predominantly TCRlow andCD24high. TCR expression is high and CD24 expressionis low on mature CD8 SP thymocytes which have beenpositively selected via H-Y TCR (Priatel et al., 2002),but this pattern is reversed on ISP thymocytes which areundergoing progression from the DN to DP stages(Egerton et al., 1990). Lack of CD69 expression, lowTCR expression and high CD24 expression indicate thatthe CD8þCD4� thymocytes of the lymphoma werenot generated through the process of TCR-mediated

Figure 4 Accelerated mortality of RasGRP1 transgenic miceresulting from coexpression of the H-Y TCR. Survival of female ormale RasGRP1 transgenic mice of the AM1268 line coexpressingH-Y TCR, compared to nontransgenic male or female miceexpressing H-Y TCR only. Circles (female RasGRP1 Tgþ /H-YTCR double transgenics) and diamonds (male RasGRP1 Tgþ /H-YTCR double transgenics) indicate deaths of individual mice, orkilling of a mouse with a large lymphoma

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positive selection, but instead were derived by transfor-mation of ISP thymocytes.

Since the H-Y TCR recognizes a male-specificantigen, in male H-Y TCRþ mice thymocytes aredeleted by negative selection as soon as they reach theDP stage (Kisielow et al., 1988). As a consequence, thereare very few DP thymocytes and no positive selectioncan occur. If lymphomagenesis is driven by theenhancement of positive selection by RasGRP1, maleRasGRP1 Tgþ /H-Y TCRþ mice would not be expectedto get thymic lymphomas. However, the death rate wasvery high in RasGRP1 Tgþ /H-Y TCRþ male mice(Figure 4). Three asymptomatic male RasGRP1 Tgþ/H-Y TCRþ mice were analysed. Of these, two had verysmall thymuses, very few DP thymocytes and no matureSP thymocytes (Figure 6a, top right and bottom leftpanels), typical of male H-Y TCRþ mice undergoing

Figure 5 Phenotype of a thymic lymphoma in a RasGRP1transgenic female mouse coexpressing H-Y TCR. The top panelsare CD4�CD8 profiles of an AM1268 RasGRP1 Tgþ /H-Y TCRfemale with a thymic lymphoma (right), compared to its H-Y TCRcontrol sister (left). The age of the mice and the total number ofthymocytes in each mouse are indicated above the dot plots. Thepercentages in each thymocyte subset (defined by boxes) are shown.The bottom four panels are overlain flow cytometry histograms ofthe CD8þ CD4� subsets (control H-Y TCR female shaded andRasGRP1 Tgþ /H-Y TCR female open). Forward light scatter is ona linear scale and H-Y TCR, CD69 and CD24 staining are on logscales

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negative selection at the DP stage. The other asympto-matic RasGRP1 Tgþ /H-Y TCRþ male also lackedmature SP thymocytes, but had increased numbers ofthymocytes including CD4þCD8� and DP thymocytes(Figure 6a, bottom right panel) which were HSAhigh andhad high light scatter (data not shown). This mouseappeared to have an early stage lymphoma comprised ofISP and DP thymocytes, despite the maintenance ofnegative selection as indicated by the absence of matureSP thymocytes.

Another RasGRP1 Tgþ /H-Y TCRþ male had a smallthymus predominantly populated by DP and CD8þ

CD4� thymocytes with high light scatter and retentionof H-Y TCR expression, while its spleen and lymphnodes were massively infiltrated with cells phenotypi-cally identical to the transformed cells in the thymus(Figure 6b, and data not shown). In this mouse, negativeselection apparently constrained the proliferation oftransformed cells within the thymus, while escape oftransformed cells from the thymus resulted in anaggressive T-cell malignancy due to lack of negativeselection in peripheral lymphoid organs.

In combination, the phenotypes of normal andtransformed thymocytes in H-Y TCRþ female and malemice indicate that RasGRP1-mediated lymphomagen-esis can originate in thymocytes which are not under-going positive selection.

TCR or pre-TCR are not required for lymphomagenesis inRasGRP1 transgenic mice

The experiments with H-Y TCR transgenic miceindicated that RasGRP1 can drive lymphomagenesisindependently of its ability to enhance TCR-dependentpositive selection, but also demonstrated that the H-YTCR transgene accelerated RasGRP1-mediated lym-phomagenesis. This transgenic TCR is ectopicallyexpressed in DN and ISP thymocytes, and providessurvival and proliferative signals that mimic pre-TCR(Von Boehmer et al., 2003). This raised the possibilitythat endogenous TCR or pre-TCR might also collabo-rate with RasGRP1 in promoting lymphomagenesis,potentially by triggering RasGRP1 activation viaPLCg1 (Ebinu et al., 2000; Bivona et al., 2003).

To directly test the requirement for either pre-TCR orTCR in RasGRP1-mediated lymphomagenesis, we

crossed the RasGRP1 transgene onto a Rag2�/� back-ground, which prevents TCR gene rearrangements. Innormal Rag2�/� mice, thymocyte development is ar-rested at the DN stage (Figure 7a, left panel). InRasGRP1 Tgþ /Rag2�/� mice, the developmental blockdue to pre-TCR deficiency was over-ridden, resulting ina 10-fold expansion of the DN population and thegeneration of ISP and DP thymocytes (Figure 7a,middle panel). This reflects the ability of RasGRP1 toprovide proliferative and developmental progressionsignals which partially compensate for pre-TCR defi-ciency (Norment et al., 2003). The RasGRP1 Tgþ /Rag2�/� mice had a high death rate (Figure 7b) due tothymic lymphomas comprised of transformed DN, ISPand DP thymocytes (example shown in Figure 7a, rightpanel). This demonstrates that the RasGRP1 transgenecan cause lymphomagenesis in the absence of abTCR,gdTCR or pre-TCR. The large numbers of DN, ISP andDP thymocytes in the RasGRP1 Tgþ /Rag2�/� miceprior to the onset of overt lymphomagenesis suggeststhat one mechanism by which deregulated RasGRP1expression can initiate lymphomagenesis is by expand-ing the pool of proliferating immature thymocytes.

Discussion

High RasGRP1 expression in most T acute lymphocyticleukemias and the presence of proviral insertions at theRasGRP1 locus in retrovirally induced murine thymiclymphomas suggested that deregulated and highRasGRP1 expression could contribute to the initiation

Figure 6 Lymphoma development despite negative selection inmale RasGRP1 transgenic mice coexpressing H-Y TCR. (a)CD4�CD8 profiles of three 293H RasGRP1 Tgþ /H-Y TCR malemice, compared to a H-Y TCR male control. All four mice areRag2�/�. The ages and total number of thymocytes in each mouseare indicated above the dot plots. The percentages in eachthymocyte subset are indicated in the boxes. (b) Top panels showCD4�CD8 profiles of a 13H RasGRP1 Tgþ /H-Y TCR malemouse, compared to a H-Y TCR male control. The middle panelsare overlain flow cytometry histograms of the DP thymocytes(control H-Y TCR male shaded and RasGRP1 Tgþ /H-Y TCRmale open). Forward light scatter is on a linear scale and TCRbstaining fluorescence intensity is on a log scale. The bottom panelsshow CD4�CD8 profiles of the spleens of this pair of mice, withtotal spleen cell numbers and percentages of CD4þ , CD8þ andCD4þCD8þ cells

Figure 7 Immature thymocyte expansion and lymphomagenesis inRasGRP1 transgenic/Rag2�/� mice. (a) CD4�CD8 profiles of oneRag2�/� mouse and two RasGRP1 Tgþ /Rag2�/� mice are shown.Ages in days, total thymocyte numbers and percentages of eachthymocyte subset (defined by quadrants) are shown. (b) Survival ofAM1268 RasGRP1 Tgþ /Rag2�/� mice, compared to nontransgenicRag2�/� mice. Diamonds indicate deaths of individual mice, orkilling of a mouse with a large lymphoma

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or progression of T-cell malignancies. The occurence ofthymic lymphomas in three independent transgenicmouse lines directly demonstrates the causative role ofRasGRP1 in the development of thymic lymphomas andderivative peripheral T-cell leukemias. The correlationbetween high transgene expression and thymocytetransformation, and our ability to measure both at thesingle cell level, allowed us to address the question ofwhether high RasGRP1 expression contributed to theinitiation versus progression of thymic lymphomas. Wefound that transformed thymocytes in very early stagelymphomas had high expression of transgenicRasGRP1, indicating that RasGRP1 contributes to theinitiation of lymphomagenesis. This presumably reflectsthe ability of RasGRP1 to stimulate signaling throughRas GTPases, as thymic lymphomas are also induced byexpression of constitutively activated mutants of N- orH-Ras (Hawley et al., 1995; Swat et al., 1996; Curtiset al., 1997), and by deregulated mutants of the Raf-1and MEK1 kinases (Iritani et al., 1997, 1999), which areactivated downstream of Ras GTPases. However, over-expression of RasGRP1 is unlikely to be sufficient toinitiate lymphomagenesis. Cooperating mutations inother genes may have to occur prior to or after thetransition to high RasGRP1 expression in order forovert transformation of thymocytes to occur. Thymo-cytes that acquired high RasGRP1 expression butlacked essential cooperating mutations might not beselectively expanded and therefore their high expressionof RasGRP1 would go unnoticed. In retrovirallyinduced T-cell lymphomas, proviral insertion at theRasGRP1 locus can be accompanied by proviralinsertions at known oncogenes, that is, Runx3, Fos,Pim1 (Kim et al., 2003). In these cases, it is very likelythat the other provirally marked oncogenes are co-operating with RasGRP1 in causing the initiation orprogression of lymphomagenesis. RasGRP1 overexpres-sion may contribute to the survival and proliferation ofthe transformed thymocytes, while additional oncogenicmutations could be required to suppress differentiation,which normally accompanies and limits RasGRP1-mediated expansion and positive selection of thymocytesat the DN, DP or SP stages (Norment et al., 2003).

In T-cell lines, RasGRP1 activation is dependent onsignaling via ligated TCR (Ebinu et al., 2000; Bivonaet al., 2003). RasGRP1 is required both for positiveselection through most TCRs and for proliferativeresponses to TCR ligation (Dower et al., 2000; Priatelet al., 2002; Layer et al., 2003). RasGRP1 transgenicmice accumulate CD8 SP thymocytes with TCR levelswhich would normally be too low to confer positiveselection, but nonetheless have a hyperactive prolifera-tive response to TCR ligation in vitro (Norment et al.,2003). In combination, these observations suggestedthat lymphomagenesis could be caused by an exagger-ated form of RasGRP1-mediated, TCR-dependentpositive selection at the DP stage, potentially combinedwith proliferative responses extending into the SP stage.We found that RasGRP1 cooperated with the positivelyselected H-Y TCR in inducing lymphomas in femalemice. However, the majority of CD8þ CD4� thymocytes

in a RasGRP1 Tgþ /H-Y TCR lymphoma were CD24high

CD69� H-Y TCRlow, indicating that they had not beenpositively selected and were probably derived from ISPthymocytes which would not be developmentally matureenough to undergo positive selection. Furthermore, theRasGRP1 transgene and the H-Y TCR were alsocooperative in promoting lymphomagenesis in nega-tively selecting male mice, in which positive selectionwas precluded and DP thymocytes were severelydepleted. Rather than implicating positive selection,these results indicated that RasGRP1-induced lympho-mas originate from DN or ISP thymocytes, prior to theexpression of ab TCR and the acquisition of suscept-ibility to positive selection.

To determine if either TCR or pre-TCR were requiredfor RasGRP1-induced lymphomagenesis, we crossed theRasGRP1 transgene onto a Rag2�/� background topreclude rearrangement of TCR genes. The RasGRP1transgenic/Rag2�/� mice did get thymic lymphomas, at arate that was markedly accelerated relative to RasGRP1transgenic/Rag2þ mice of the same line. In contrast toRag2�/� mice in which thymocyte development isarrested at the DN stage due to pre-TCR deficiency,the RasGRP1 transgenic/Rag2�/� mice had large num-bers of DN, ISP and DP thymocytes, even prior to theonset of lymphomagenesis. This suggests that deregu-lated expression of RasGRP1 contributes to the initia-tion of lymphomas by triggering proliferation of DNand ISP thymocytes, thus expanding the pool ofimmature thymocytes that are susceptible to transfor-mation. This mechanism also explains the ability of theH-Y transgenic TCR to cooperate with RasGRP1 incausing lymphomas. Abnormally early expression of thetransgenic H-Y TCR results in excessive accumulationof DN thymocytes (Von Boehmer et al., 2003),apparently due to perturbed gd versus ab lineagecommitment (Bruno et al., 1996; Terrence et al., 2000;Erman et al., 2002). When combined with the ability ofderegulated expression of RasGRP1 to stimulate DNand ISP proliferation, this effect of the H-Y TCR wouldbe expected to increase the probability of lymphomainitiation.

Materials and methods

Generation and breeding of RasGRP1 transgenic mice

The transgenic expression vector p1017C was derived fromp1017D (Norment et al., 2003) by inserting the XbaI fragmentof the IgH enhancer upstream of the lck promoter, exactly asdescribed (Iritani et al., 1997). The N-terminally HA-taggedRasGRP1 cDNA was inserted into p1017C at the MluI andSalI sites between the b-globin intron and the polyadenylationsignal of the human growth hormone gene. The 13H and293H founder mice were obtained by microinjecting theNotI fragment from p1017C/HA-RasGRP1 into (C57BL/6�DBA)�C57BL/6 F1 embryos, and the transgenic lineswere established by breeding to C57BL/6. The generation ofAM1268 transgenic mice was described previously (Normentet al., 2003).

RasGRP1 transgenic mice were bred with Rag2�/� C57BL/6mice (Shinkai et al., 1992). RasGRP1 Tgþ/Rag2þ /� progeny

Oncogene

were then crossed with the Rag2�/� mice to obtain mixedlitters including Rag2�/� and RasGRP1 Tgþ /Rag2�/� mice.RasGRP1 transgenic mice were crossed to H-Y TCRþ /þ/Rag2�/� C57BL/6 mice to produce mixed litters includingRasGRP1 Tgþ /H-Y TCRþ /�/Rag2þ /� and H-Y TCRþ /�/Rag2þ /� mice. RasGRP1 Tgþ /H-Y TCRþ /�/Rag2�/� micewere produced from crosses of RasGRP1 Tgþ /Rag2�/� and H-Y TCRþ /þ/Rag2�/�. All H-Y TCR mice were on a H-2Db

background. Rag2�/� and Rag2�/� H-Y-TCR C57BL/6 micewere purchased from Taconic Farms (Germantown, NY,USA). Mice were housed under pathogen-free conditions.

Flow cytometry analysis

The following antibody conjugates were from BD Pharmingen(San Diego, CA, USA): anti-CD4-CyChrome, anti-CD4-APC,anti-CD8a-APC, anti-CD8a-PE, anti-TCRb-FITC, anti-TCR-Vb8-FITC, anti-CD24-FITC, anti-CD69-FITC, anti-CD25-FITC, anti-CD5-FITC, anti-CD62L-FITC. H-Y TCR expressionwas detected with FITC-T3.70 (eBioscience, San Diego, CA,USA). After staining with antibodies and propidium iodide, cellswere analysed using a FACScalibur flow cytometer and CellQuestsoftware (Becton Dickinson, San Jose, CA, USA). In thedisplayed analyses, nonviable cells which were propidium iodidepositive or had very low forward light scatter were gated out.

Intracellular staining/flow cytometry detection of RasGRP1transgenic protein

Thymocytes were isolated, rinsed once with HBSS/2% FBSand incubated with anti-CD4-APC and anti-CD8-PE for30min. Thymocytes were then rinsed twice with HBSS/2%FBS and fixed with cytofix/cytoperm solution (BD Pharmin-gen) for 20 min. Fixed thymocytes were rinsed twice with 1�cytoperm/wash solution and incubated with FITC-conjugatedanti-HA antibody (Covance Research Products, Berkeley, CA,USA) for 30 min. Stained thymocytes were rinsed twice with1� cytoperm/wash and suspended in HBSS/2% FBS andanalysed by flow cytometry.

Analysis of TCR-Vb8 to total TCRb ratios

Thymocytes were stained with anti-CD4-CyChrome and anti-CD8a-APC, in conjunction with either anti-TCRb-FITC oranti-TCR-Vb8-FITC. Transformed thymocyte populationswere gated by CD4, CD8 and/or by high light scatter asneeded to segregate them from nontransformed thymocytes.The proportion of gated cells expressing TCR-Vb8 or TCRbwere quantified by histogram analysis.

Cell cycle analysis

Thymocytes were isolated, rinsed in HBSS/2% FBS and fixedwith ice-cold 70% ethanol and left at 41C overnight. Fixedthymocytes were rinsed twice with HBSS and incubated withpropidium iodide (5 mg/ml) and RNase A (200mg/ml) at 41 forat least 4 h, and analysed by flow cytometry.

Culture of lymphoma cells and derivation of cell lines

Normal or lymphomic thymuses were disaggregated by forcingthrough a nylon mesh and then put in culture in RPMI, 10%FBS. Cultures were maintained by removing medium and excessnonadherent cells every 3 days and replacing with fresh medium.After 7–14 days of culture, cells derived from lymphomas weremaintained as pools or cloned by dilution and expanded as celllines. In contrast to the thymic lymphomas, no proliferation ofnormal RasGRP1 transgenic thymuses occurred in culture, andno cell lines could be established.

Acknowledgements

We are grateful to Rewa Grewal, Carolyn Bateman, TraceySutcliffe and others at the BCCRC Joint Animal Facility fortheir contributions to generating and maintaining our mice.Nastaran Mohammadi and Heather Kirk provided excellenttechnical assistance with the mouse genotyping. This work wassupported by operating grants from the Canadian Institutes ofHealth Research and the Cancer Research Society to RJK, andby a CIHR doctoral research award to MBK.

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Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors

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