The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Norment, A. M.
Right arrow Articles by Kay, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Norment, A. M.
Right arrow Articles by Kay, R. J.
The Journal of Immunology, 2003, 170: 1141-1149.
Copyright © 2003 by The American Association of Immunologists

Transgenic Expression of RasGRP1 Induces the Maturation of Double-Negative Thymocytes and Enhances the Production of CD8 Single-Positive Thymocytes 1

Anne M. Norment2,*, Lisa Y. Bogatzki3,*,{dagger}, Mark Klinger{ddagger}, Ethan W. Ojala4,*,{dagger}, Michael J. Bevan*,{dagger} and Robert J. Kay5,{ddagger}

* Department of Immunology and {dagger} Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195; {ddagger} Terry Fox Laboratory, British Columbia Cancer Agency, and Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RasGRP1 is a guanine nucleotide exchange factor for Ras that is required for the efficient production of both CD4 and CD8 single-positive thymocytes. We found that RasGRP1 expression is rapidly up-regulated in double-negative thymocytes following pre-TCR ligation. Transgenic overexpression of RasGRP1 compensated for deficient pre-TCR signaling in vivo, enabling recombinase-activating gene 2-/- double-negative thymocytes to mature to the double-positive stage. RasGRP1 transgenic mice had a 4-fold increase in CD8 single-positive thymocytes, most of which had atypically low levels of CD3. The RasGRP1 transgene lowered the threshold of TCR signaling needed to initiate proliferation of single-positive thymocytes, with this effect being particularly evident among CD8 single-positive cells. In 3-day cultures, TCR stimulation via anti-CD3 caused a 10-fold increase in the ratio of CD8 to CD4 thymocytes among RasGRP1 transgenic vs nontransgenic thymocytes. These results demonstrate that in addition to driving the double-negative to double-positive transition, increased expression of RasGRP1 selectively increases CD8 single-positive thymocyte numbers and enhances their responsiveness to TCR signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During thymocyte development, {alpha}{beta} T-lineage cells pass through an ordered series of differentiative steps in response to pre-TCR- and TCR{alpha}{beta}-derived signals. In immature CD4-CD8- (double-negative (DN)6) thymocytes, assembly of the TCR {beta}-chain into a functional pre-TCR complex delivers signals that stimulate a rapid burst in cellular proliferation in tandem with differentiation to the CD4+CD8+ (double-positive (DP)) stage (1, 2). Pre-TCR signaling also induces transcription and rearrangement at the TCR{alpha} locus, resulting in formation of a TCR{alpha}{beta} heterodimer as thymocytes reach the DP stage (3). Signaling from the pre-TCR appears to be ligand independent, potentially initiated solely by the complete assembly of the pre-TCR complex following TCR{beta} expression (1). In contrast, TCR{alpha}{beta} signaling is induced by binding to MHC/peptide complexes and occurs in conjunction with engagement of the MHC class-specific coreceptor CD4 or CD8. The affinities of the MHC/peptide-TCR{alpha}{beta} interactions direct the further development of DP thymocytes, with strong affinities triggering deletion, minimal affinities leading to death by neglect, and intermediate affinities promoting survival and differentiation to either the CD4+CD8- (CD4 single-positive (SP)) or the CD4-CD8+ (CD8SP) lineage (4).

The ERK1 and ERK2 kinases have been implicated as critical downstream mediators of the effects of pre-TCR and TCR signaling on thymocyte development (4, 5, 6, 7). Increased ERK activity accompanies activation of the pre-TCR by CD3 ligation or by initiation of expression of TCR{beta} in DN cells (8, 9). The DN to DP transition in thymocyte development is enhanced by the expression of activated forms of H-Ras or Raf-1 (10, 11, 12) and in thymic organ cultures is impeded by a dominant negative form of the ERK-activating kinase MEK1 (mitogen-activated protein kinase kinase) (9), indicating that ERK activation, stimulated by the formation of functional pre-TCR, is what drives this step in differentiation. At the DP stage, effective positive selection of thymocytes expressing MHC-interactive TCR is impeded by inhibition of MEK1 or loss of ERK activation via TCR signaling (13, 14, 15, 16, 17), while hyperactivation of MEK can cause positive selection in the absence of functional TCR (15). Activated Raf-1 can increase the efficiency of positive selection, while dominant negative Raf-1 suppresses positive selection (18). Negative selection of DP thymocytes induced by CD3 ligation or TCR-specific peptides can be suppressed by MEK inhibition (19, 20), although in other experimental systems negative selection can proceed despite MEK inhibition (13, 14, 15). The differentiation of DP thymocytes to CD4 SP is impeded when MEK is inhibited, while differentiation to the CD8 SP lineage is either unaffected or aberrantly promoted, e.g., in the absence of MHC class I on APC or in the absence of an MHC class I-restricted TCR on the DP thymocytes (20, 21, 22, 23).

One mechanism for activation of the Raf-1/MEK1/ERK cascade of kinases is through the Ras GTPases (6). Expression of a constitutively active V12 form of H-Ras enables the development of DP thymocytes from DN thymocytes lacking the TCR{beta} component of pre-TCR (10, 11). Activated H-Ras does not provide equivalence to all signals derived from pre-TCR or TCR, as the RAG-deficient DP thymocytes expressing V12 H-Ras failed to undergo allelic exclusion at the TCR{beta} locus and failed to develop further into SP thymocytes (10, 11). In DP thymocytes the expression of N17 H-Ras, a dominant negative mutant that sequesters Ras-specific exchange factors (24), impedes positive selection while having no effect on negative selection (25). The evidence is still tenuous for Ras being the principal upstream transducer of signals leading from pre-TCR or TCR to ERK in thymocytes. Loss of one allele of Grb2, an adaptor involved in recruiting the Ras-specific exchange factor Sos to receptors, reduced Ras activation in total thymocytes in response to TCR cross-linking, but ERK activation in these circumstances was unaffected (26). Expression of N17 H-Ras did suppress ERK activation via TCR/CD3 cross-linking (25, 26), indicating that at least one type of Ras exchange factor, but perhaps not Sos, is involved in TCR to ERK signaling in SP thymocytes.

RasGRP1 (also known as RasGRP or CalDAG-GEFII) is an exchange factor that is expressed in the thymus and can activate H-Ras, K-Ras, and N-Ras and the Ras-related GTPases R-Ras and TC21 (27, 28, 29, 30). RasGRP1 is activated by recruitment to diacylglycerol or phorbol ester-enriched membranes (27, 28, 30, 31) and thus can provide a route to Ras activation downstream of any phospholipase C-coupled receptor, including TCR or pre-TCR. In the Jurkat T cell line, a high proportion of Ras activation in response to TCR cross-linking is mediated by RasGRP1, and overexpression of RasGRP1 in these cells prolongs the ERK activation response to TCR cross-linking (31). An inactivating mutation in both alleles of RasGRP1 causes a 10-fold reduction in CD4 SP thymocytes and a 4-fold reduction in CD8 SP thymocytes, with the numbers of DP thymocytes being modestly reduced and DN thymocyte numbers being increased to a variable extent (32). RasGRP1 is thus implicated as a critical mediator of thymocyte development, particularly in processes required for SP thymocyte production.

We found RasGRP1 in a screen for genes whose expression is induced in recombinase-activating gene 2 (RAG-2)-/- DN thymocytes following ligation of CD3. The up-regulation of RasGRP1 via pre-TCR stimulation, the modest accumulation of DN thymocytes in RasGRP1-deficient mice, and the ability of activated Ras to drive the DN to DP transition in RAG-deficient mice (10, 11) suggested that RasGRP1 could contribute to the induction of DN thymocyte maturation that occurs in response to pre-TCR signaling. To test this hypothesis we produced mice in which RasGRP1 is elevated in DN and other thymocytes via transgene expression. The RasGRP1 transgenic DN thymocytes were able to mature into DP thymocytes in the absence of TCR{beta}, indicating that RasGRP1 may be contributing to pre-TCR-induced signals that drive the DN to DP transition. In addition to its effects on the DN to DP transition, the RasGRP1 transgene conferred a large increase in the output of CD8 SP thymocytes characterized by low CD3 levels and selectively enhanced the proliferation of CD8 SP thymocytes in response to TCR cross-linking. These results provide evidence that variation in RasGRP1 expression can be a significant determinant of pre-TCR- and TCR-mediated thymocyte development and can selectively influence the CD8 lineage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Flow cytometric analysis

The following mAb conjugates were obtained from BD PharMingen (San Diego, CA): biotinylated mAb to murine CD4, CD8, CD5, and CD3{epsilon}; anti-CD44-PE; anti-CD25-FITC; anti-CD4-CyChrome; anti-CD8{alpha}-allophycocyanin; anti-CD8{alpha}-PE; anti-CD24-FITC (HSA); anti-CD3{epsilon}-PE; anti-CD69-FITC; and anti-CD69-PE. PE- and Tri-color-conjugated streptavidin and anti-CD4-Tri-color were obtained from Caltag Laboratories (Burlingame, CA). Following mAb staining, cells were fixed in 1% paraformaldehyde and analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences, San Jose, CA).

Northern blot analysis and RT-PCR

C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). RAG-2-/- mice (originally from Dr. F. Alt) were bred at the University of Washington facility. For some experiments additional RAG-2-/- mice were purchased from Taconic Farms. Total RNA was isolated from thymi of 4-wk-old PBS- or anti-CD3{epsilon}-injected RAG-2-/- mice or tissues of adult C57BL/6 mice, separated on a 1% agarose formaldehyde gel, and analyzed by Northern blot analysis as previously described (33). Blots were probed using a full-length murine RasGRP1 cDNA probe (30) or elongation factor-1{alpha} as a loading control. For RT-PCR analysis, thymocytes from 6- to 8-wk-old C57BL/6 mice were sorted using a FACStar Plus flow cytometer (BD Biosciences) into DP, CD4 SP, and CD8 SP fractions as previously described (34). C57BL/6 thymocytes were enriched for DN cells using biotinylated mAb to murine CD4 (RM4-5), CD8 (53-6.7), and CD3{epsilon} (145-2C11), followed by depletion using streptavidin-coated magnetic Dynabeads (Dynal, Oslo, Norway) according to the manufacturer’s instructions. The remaining cells were stained using anti-CD44-PE, anti-CD25-FITC, and streptavidin-Tri-color and then sorted to obtain individual CD44/CD25 triple-negative subsets. RNA and cDNA were prepared from sorted cells and normalized according to hypoxanthine phosphoribosyltransferase (HPRT) expression as previously described (33), after 30 PCR cycles using the primers HPRT 7/8 and 9 (34). PCR of RasGRP1 was performed for 30 cycles (94°C for 1 min, 60°C for 1 min, 72°C for 1 min) using the primers RasGRP 6 (5'-CGGATCATCATCTCCTCAGCC-3') and RasGRP 8 (5'-CAGTCATCTCGCCCAGAACC-3'). PCR products were visualized by Southern blotting using an HPRT probe generated using the primers listed above or an internal EcoRV-BglII RasGRP1 cDNA fragment.

Generation of RasGRP1 transgenic mice and breeding to RAG-2-/- mice

The transgenic expression vector p1017D was derived from the lck proximal promoter vector p1017 (35) by removing multiple introns of the human growth hormone gene (retaining the growth hormone polyadenylation signal, bp 1361–2154; GenBank accession no. M13438) and inserting the second intron of the rabbit {beta}-globin gene (bp 551-1242; GenBank accession no. V00878) downstream of the lck promoter sequence. The RasGRP1-p1017D transgene expression construct was generated by adding three consecutive hemagglutinin (HA) epitope tags to the 3' end of the full-length murine RasGRP1 cDNA (30). The encoded protein has the C-terminal sequence GDSAGLKSTEAYPYDVPDYASGSYPYDVPDYASGSYPYDVPDYASG. The natural C-terminal sequence of RasGRP1 is GDSA. The RasGRP1/HA cDNA was inserted into p1017D at the MluI and SalI sites between the {beta}-globin intron and the growth hormone polyadenylation signal. RasGRP1-HA founder animals were obtained by microinjecting the NotI fragment from p1017D-RasGRP1-HA into (C57BL/6 x DBA) x C57BL/6 F1 embryos and were bred to C57BL/6 mice to establish lines. Transgene positive mice were identified by PCR of tail DNA using the RasGRP 6 and 8 primers. RasGRP1 transgenic mice (AM 1268 line) were bred with RAG-2-/- mice (36). RasGRP1 transgenic/RAG-2+/- progeny were then interbred to obtain RasGRP1 transgenic/RAG-2-/- mice for analysis. The RAG-2 genotype of mice was identified by PCR of tail DNA using PCR primers for RAG-2 (5'-CCAGCTGATAACCACCCACAA-3' and 5'-GTATAGTCGAGGGAAAAGCAT-3') and neomycin phosphotransferase (5-TGGGATCGGCCATTGAACAAG-3' and 5'-CACGGGTAGCCAACGCTATGT-3'). All mice were housed under specific pathogen-free conditions.

Western blot analysis

Thymocytes of anti-CD3{epsilon}-treated RAG-2-/- mice (33) or thymocytes and splenocytes of 4- to 8-wk-old RasGRP1 transgenic mice or nontransgenic littermates were harvested by disaggregation through a wire mesh. Cells were lysed at 108 cells/ml (for RAG-2-/- cells) or 2 x 108 cells/ml at 4°C for 30 min in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, 50 mM iodoacetamide, 50 mM NaF, 50 mM {beta}-glycerol, 10 mM EDTA, 1 mM Na3V04, 1 mM PMSF, 10 µg/ml of aprotinin, and 10 µg/ml of leupeptin, followed by centrifugation to remove the insoluble pellet. For RAG-2-/- protein lysates, 106 cell equivalents were separated by SDS-PAGE on a 12% gel, transferred to an Optitran nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and immunoblotted using the following Ab: monoclonal mouse anti-RasGRP, rabbit polyclonal anti-Sos 1/2, or goat polyclonal anti-ERK2 (all from Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit polyclonal anti-phospho-p44/42 mitogen-activated protein kinase (ERK; New England Biolabs, Beverly, MA). For RAG-2-/- protein lysates, immunoblotting was performed in Tris-buffered saline/0.1% Tween 20 with 5% nonfat dry milk according to the manufacturer’s protocol for anti-phospho-p44/42 mitogen-activated protein kinase (New England Biolabs). For detection of RasGRP1 in transgenic thymocytes or splenocytes, lysates from 4 x 106 cell equivalents were resolved on an 8% gel and immunoblotted with mouse anti-RasGRP or mouse mAb HA.11 (Covance/Berkeley Antibody Co., Richmond, CA) in PBS/0.05% Tween 20 with 5% nonfat dry milk. Bound Abs were detected with HRP-conjugated secondary Abs (Santa Cruz Biotechnology) and ECL according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Little Chalfont, U.K.). Blots were stripped according to the protocol described by Amersham.

Intracellular staining/flow cytometric detection of RasGRP1 transgenic protein

Thymocytes from transgenic and nontransgenic littermate mice from AM1198 and AM1268 lines between 6 and 8 wk of age were isolated, rinsed once with HBSS/2% FBS, and incubated with anti-CD4-APC and anti-CD8-PE for 30 min. Thymocytes were then rinsed twice with HBSS/2% FBS and fixed with Cytofix/Cytoperm solution (BD PharMingen) for 20 min. Fixed thymocytes were rinsed twice with 1x Cytoperm/wash solution and incubated with FITC-conjugated anti-HA Ab for 30 min. Stained thymocytes were rinsed twice with 1x Cytoperm/wash, suspended in HBSS/2% FBS, and analyzed by flow cytometry.

5-Bromodeoxyuridine (BrdU) incorporation and proliferation assays

RasGRP1 transgenic or nontransgenic littermate mice were given two i.p. injections of 1 mg of BrdU, given 2 h apart. Twenty-four hours later, thymocytes were harvested and stained using anti-CD4-Tri-color and anti-CD8{alpha}-PE, followed by staining with FITC-conjugated anti-BrdU (BD Biosciences) essentially as previously described (34). For analysis of in vitro proliferation using CFSE, thymocytes (108/ml) were labeled with 2.5 µM CFDA in PBS (Molecular Probes, Eugene, OR) for 10 min at 37°C. Cells were washed three times with ice-cold RMPI 1640 medium, recounted, and incubated in triplicate in 96-well plates at 2 x 105 cells/200 µl of RPMI 1640 containing 10% FBS (HyClone, Logan, UT), 2 mM glutamine, 25 mM HEPES, 50 µM 2-ME, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. For anti-CD3{epsilon} stimulation, wells were previously incubated overnight at 4°C with 50 µg/ml rabbit anti-hamster IgG (Sigma-Aldrich, St. Louis, MO) in PBS and washed with PBS, followed by affinity-purified anti-CD3{epsilon} (33), at the indicated concentration in PBS for 2 h at 37°C. After 48 or 72 h cells were stained using anti-CD4-Cy-Chrome and anti-CD8{alpha}-APC and were analyzed by flow cytometry. The average number of cell divisions was derived from calculation of the area of each peak of CFSE fluorescence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RasGRP1 expression is up-regulated at the DN to DP transition during thymocyte development

To determine the pattern of RasGRP1 expression during thymocyte development, we performed quantitative RT-PCR analysis using cDNA from sorted thymocyte subsets (Fig. 1). RasGRP1 mRNA was present at low amounts in total DN thymocytes as well as in the CD25+CD44- subset of DN thymocytes that are dependent on pre-TCR function for their further development (36). DP thymocytes contained ~10-fold higher levels of RasGRP1 mRNA compared with CD25+CD44- DN thymocytes. The levels of RasGRP1 transcripts declined slightly in both lineages of SP thymocytes, but remained well above those found in DN thymocytes.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 1. RT-PCR analysis of RasGRP1 mRNA expression in thymocyte subsets. Three-fold serial dilutions of cDNA from sorted subsets of thymocytes (total DN, CD44-CD25+ DN, DP, CD4SP, and CD8SP) or total thymocytes (total) were analyzed by RT-PCR using primers for RasGRP1 (top panel) or HPRT as a normalization control (bottom panel). Reaction products were visualized by Southern blotting. The relative quantity of RasGRP1 transcripts in each thymocyte subset was determined by estimation from the dilution series of the PCR reaction, setting the DN subset at 1, and then normalizing to the relative amounts of HPRT signal for each subset. DN cells were sorted as CD4- CD8- CD3-. The purity of the sorted populations was verified by flow cytometry and by RT-PCR for CD4 and CD8.

 
To examine RasGRP1 expression during pre-TCR-dependent thymocyte maturation, we used RAG-2-/- mice in which thymocyte development is arrested at the DN stage due to failure to form the TCR{beta} component of the pre-TCR (36, 37). Injection of anti-CD3{epsilon} mAb into RAG-2-/- mice triggers signaling by ligating the CD3 components of the residual pre-TCR complex, and despite the absence of TCR{beta}, this is sufficient to induce synchronous differentiation of DN thymocytes into DP thymocytes (33, 38, 39). As measured by Northern blot, RasGRP1 mRNA was present at very low levels in unstimulated RAG-2-/- thymocytes (which are >99% DN thymocytes, predominantly at the CD25+CD44- stage) and was strongly up-regulated within 7 h of anti-CD3 injection (Fig. 2A). At this time point, the majority of RAG-2-/- thymocytes have received an activation signal, as measured by elevated CD69 expression (data not shown). RasGRP1 mRNA expression remained elevated up to 72 h post-anti-CD3 treatment at levels comparable to those in total thymocytes from wild-type mice (which are ~85% DP thymocytes). Thus, RasGRP1 mRNA is rapidly up-regulated by pre-TCR signaling, and the high levels of RasGRP1 expression are maintained as the DN thymocytes differentiate.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 2. Induction of RasGRP1 expression by CD3 cross-linking. A, Northern blot analysis of RasGRP1 expression in anti-CD3-stimulated RAG-2-/- thymocytes. RNA was isolated from pooled total thymocytes of RAG-2-/- mice after the indicated number of hours postinjection with anti-CD3{epsilon} mAb. For comparison, RNA was also isolated from total thymocytes of wild-type mice (RAG-2+/+ thymus). The blot was successively hybridized with probes for RasGRP1 (top panel) and elongation factor-1{alpha} (bottom panel) as a loading control. B, Immunoblot analysis of RasGRP1 expression in thymocytes from anti-CD3{epsilon}-stimulated RAG-2-/- mice. Protein lysates were obtained from thymocytes from RAG-2-/- mice after the indicated number of hours postinjection with anti-CD3{epsilon} mAb. Following electrophoresis and transfer, the membrane was successively immunoblotted with Abs to RasGRP1 (top panel), Sos-1 (second panel), phospho-ERK1/2 (p-ERK; third panel), or total ERK1/2 as a loading control (ERK; bottom panel).

 
Western blot analysis of lysates from RAG-2-/- thymocytes showed that RasGRP1 protein levels increased within 4 h of anti-CD3 injection, with a continuing increase in levels over the 24-h time course (Fig. 2B). ERK1/2 activation was also observed at the 4 h point, although the subsequent accumulation of RasGRP1 was not accompanied by a further increase in ERK1/2 activation. In contrast to RasGRP1, the Ras exchange factor Sos-1 was expressed in unstimulated DN thymocytes, and its levels did not change in response to anti-CD3 injection (Fig. 2B).

Generation of RasGRP1 transgenic mice

The induction of its expression by pre-TCR ligation suggested a role for RasGRP1 in the processes that drive the DN to DP transition, in addition to its previously identified role in the DP to SP transition. To determine the functional effects of increased expression of RasGRP1, we generated mice carrying a RasGRP1 transgene with the following components: the proximal lck promoter that provides expression in thymocytes (35), a rabbit {beta}-globin gene-derived intron, a cDNA encoding RasGRP1 with a C-terminal HA tag, and a polyadenylation signal derived from the human growth hormone gene. Most transgenic lines produced with this p1017D vector had high expression of the RasGRP1 cDNA in thymocytes. In contrast, no expression was observed in multiple transgenic lines derived with the p1017 vector (35), which has a series of introns positioned downstream of the inserted cDNA. Introns within a 3'-untranslated region can promote transcript degradation (40).

Two RasGRP1 transgenic lines, AM1198 and AM1268, were chosen for further study. Immunoblot analysis with anti-HA Ab showed the expression of transgenic RasGRP1 in each of the DP, CD4 SP, and CD8 SP thymocyte subsets of the AM1268 line (Fig. 3A). Reprobing the blots with an anti-RasGRP1 Ab showed that the transgenic and endogenous RasGRP1 (distinguished by the small size shift imposed by the HA tag) were expressed at roughly equivalent levels (Fig. 3A), indicating an approximate doubling of RasGRP1 levels in the transgenic thymocytes. Combined staining of thymocytes with anti-CD4, anti-CD8, and anti-HA Abs (the latter after cell permeabilization), followed by flow cytometric analysis, showed that HA-tagged RasGRP1 was expressed at equivalent levels in each of the DN, DP, CD4 SP, and CD8 SP subsets in the AM1268 transgenic line (Fig. 3B). In the AM1198 line, transgenic RasGRP1 expression was present in most CD8 SP, absent in DP, and present in a small proportion of CD4 SP and DN thymocytes. We have observed similar variation in expression among discreet subsets of thymocytes for several different transgenes, in p1017D and other vectors. This presumably reflects position-effect variegation affecting transcriptional efficiency in a cell type-specific manner.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. Transgenic expression of RasGRP1 in thymocytes. A, Western blot analysis of transgenic and total RasGRP1 protein in sorted thymocyte subsets. Protein lysates from sorted DP, CD4 SP, or CD8 SP thymocyte subsets from transgenic (Tg+) or age-matched nontransgenic (Tg-) mice of the AM1268 line were resolved on a denaturing 10% polyacrylamide gel. The same blot was successively immunoblotted with anti-RasGRP1 (top panel) or anti-HA (bottom panel). Arrows indicate bands that are superimposable when the membrane was immunoblotted with anti-RasGRP1 or anti-HA. The HA-tagged transgenic RasGRP1 protein migrated slightly slower than the endogenous RasGRP1 on SDS-PAGE. B, Flow cytometric analysis of transgenic RasGRP1 protein expression in thymocyte subsets. Thymocytes from transgenic (Tg+) and nontransgenic (Tg-) littermate mice from 1198 and 1268 RasGRP1 lines at 6 and 8 wk of age were treated with anti-CD4-APC and anti-CD8-PE, fixed, treated with anti-HA-FITC, and then analyzed by flow cytometry. Histogram overlays show log-scale anti-HA fluorescence intensity of thymocyte subsets, with black lines for transgenic and gray lines for nontransgenic. Numbers represent the mean fluorescence intensity of each histogram.

 
The RasGRP1 transgene drives the DN to DP transition in RAG-2-/- mice

As demonstrated above, RasGRP1 expression is rapidly up-regulated following pre-TCR signaling initiation. To determine whether increased RasGRP1 expression contributes to the differentiation signal provided by pre-TCR signaling, we tested the ability of the RasGRP1 transgene to over-ride the DN to DP differentiation block that occurs in RAG-2-/- mice. Because of their inability to express the TCR {beta}-chain of the pre-TCR complex, RAG-2-/- mice have virtually no DP thymocytes (Table I). However, among 13 RAG-2-/- mice expressing the RasGRP1 transgene, eight had significantly elevated percentages of DP thymocytes (an average of 34% DP among these eight mice vs 0.16% DP in control RAG-2-/- mice; Table I). Representative flow cytometric profiles for RAG-2-/- vs RasGRP1 transgene+/RAG-2-/- thymi are shown in Fig. 4. The total number of thymocytes was also increased in the eight RasGRP1 transgene+/RAG-2-/- mice with elevated DPs to an average of 20 x 106 vs 1.9 x 106 in control RAG-2-/- mice (Table I). As a result, these mice had a considerable increase in the number of DP cells (average of 9 x 106 vs 0.003 x 106 in RAG-2-/- mice lacking the RasGRP1 transgene). Therefore, the RasGRP1 transgene can compensate for the absence of TCR{beta} in driving the differentiation of DN thymocytes to the DP stage.


View this table:
[in this window]
[in a new window]
  		Table I. Numbers of DP and DN thymocytes in RAG-2-/- mice with or without the RasGRP1 transgenea

 


View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 4. Flow cytometric analysis of thymocytes from RAG-2-/- (bottom panel) and RasGRP1 Tg+/RAG-2-/- mice from the same (middle panel) or different litters (top panel). The percentages in each population are shown. The absolute numbers of thymocytes in these mice are listed in Table I.

 
RasGRP1 transgenic mice have increased numbers of CD8 SP thymocytes

In normal (RAG-2+/+) mice, the RasGRP1 transgene did not affect DP or DN thymocyte numbers or proportions. Instead, the major change in thymocyte populations occurred in the CD8 SP subset, which was increased >3-fold above normal levels in both 1268 and 1198 RasGRP1 transgenic mice (Table II). Representative flow cytometry profiles comparing RasGRP1 transgenic vs nontransgenic thymocyte populations are shown in Fig. 5A. The CD8 SP thymocytes in the transgenic mice all express TCR{alpha}{beta} (data not shown). To evaluate the maturity of the CD8 SP thymocytes in RasGRP1 transgenic mice, we measured their expression of CD3, HSA (CD24), CD5, CD69, and CD62L. Data in Figs. 5B and 6 are from mice of the 1268 line, but nearly identical profiles were consistently observed for mice of the 1198 line (data not shown). CD3 levels increase as thymocytes mature from the DP through the SP stage. SP thymocytes can be further subdivided, with the more mature cells bearing lower levels of HSA (41). CD8 SP thymocytes of RasGRP1 transgenic mice demonstrated an unusual phenotype, in that a high proportion expressed low levels of both CD3 and HSA (Fig. 5B). CD5 expression increases after positive selection and has been reported to correlate with TCR signaling intensity (42). Transgenic CD8 SP thymocytes showed a slight, but consistent, reduction in cell surface CD5 (Fig. 5B). Although CD69 is reported to be up-regulated by Ras activation (43), CD8 SP transgenic thymocytes had distinctly lower CD69 surface expression relative to nontransgenic littermate controls (Fig. 6). The CD69- CD62L+ phenotype of the majority of transgenic CD8 SP cells further indicates that they are relatively mature (44, 45). In contrast to CD8 SP cells, the CD4 SP thymocytes of RasGRP1 transgenic mice were equivalent to those of normal mice in terms of CD3, HSA, CD69, and CD62L expression (Figs. 5B and 6).


View this table:
[in this window]
[in a new window]
  		Table II. Percentages of SP thymocytes in RasGRP1 transgenic vs nontransgenic micea

 


View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 5. Flow cytometric analysis of thymocytes from RasGRP1 transgenic mice. A, CD4/CD8 profiles comparing thymocytes from littermate transgenic (Tg+) or nontransgenic (Tg-) mice of the AM1268 and AM1198 lines are shown. Boxes indicate the gates used to analyze the expression of surface markers on CD8 SP or CD4 SP subsets in B. The percentages of live gated cells in each population are shown. B, Profiles of CD3, HSA, or CD5 expression for RasGRP1 transgenic (thick line) or nontransgenic littermate (thin line) are shown for CD8 SP or CD4 SP thymocytes. The mean fluorescence intensity for transgenic (Tg+) or nontransgenic (Tg-) cells falling within the indicated region (horizontal bar) are given in the upper left corner of each histogram. For HSA, the region marker was placed to evaluate expression in the more mature (HSA low) SP subset.

 


View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 6. Analysis of maturation and selection markers on SP thymocytes from littermate transgenic (Tg+) or nontransgenic (Tg-) mice of the AM1268 line. The numbers are the percentages of cells in each quadrant.

 
While BrdU incorporation in DN, DP, and CD4 SP subsets was not significantly affected by the RasGRP1 transgene, the CD8 SP cells from transgenic mice incorporated 3-fold less BrdU than did CD8 SP cells of nontransgenic littermates (Table III). The HSAlow CD3low CD8 SP cells in the transgenics had disproportionately few cells with high forward scatter relative to the same cells in nontransgenics or to CD3high HSAhigh CD8SPs in either transgenics or nontransgenics (data not shown), indicating that the excess of nonproliferative CD8 SP cells in the transgenics are in the HSAlow CD3low subset. Thus, the surplus of CD8 SP thymocytes in RasGRP1 transgenic mice appears to be predominantly composed of nonproliferative HSAlow CD69- CD62L+ cells, which thereby resemble mature SP thymocytes, but have anomalously low levels of CD3.


View this table:
[in this window]
[in a new window]
  		Table III. BrdU incorporation in thymocyte subsetsa

 
Increased RasGRP1 expression promotes selective proliferation of CD8 SP thymocytes in response to TCR cross-linking

As a functional test of the effects of the RasGRP1 transgene on the abilities of CD8 SP vs CD4 SP thymocytes to respond to TCR signaling, we monitored the viable proportions of thymocyte subsets during in vitro culture in the absence or the presence of stimulation via plate-bound anti-CD3. Culture without anti-CD3 led to the death of most thymocytes over 72 h. Under these nonstimulatory conditions, the CD8 SP:CD4 SP ratio was reduced 3-fold in the transgenic thymocyte cultures and 2-fold in the nontransgenic thymocyte cultures (Fig. 7A). Therefore, the increased proportion of CD8 SPs in the transgenic mice is not due to a general resistance to apoptosis, being selectively conferred on CD8 SP thymocytes by increased RasGRP1 expression.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 7. Thymocyte responses to TCR cross-linking. A, Proportions of thymocyte subsets in cultures with and without stimulation via anti-CD3. Transgenic or nontransgenic thymocytes of the AM1268 line were cultured for the indicated times in the presence or the absence of anti-CD3{epsilon} coated onto plates at 0.5 µg/ml, then stained with anti-CD4- and anti-CD8, and analyzed by flow cytometry. The percentages of live gated cells in each population are shown along with the derived ratios of CD8 SP to CD4 SP thymocytes. B, Proliferation of thymocytes in the absence or the presence of anti-CD3 stimulation. Thymocytes from transgenic or nontransgenic mice of the AM1268 line (same experiment as that shown in A) were labeled with CFSE and then stimulated for 72 h in plates coated with anti-CD3{epsilon} at 0 (nil), 0.5 (high), or 0.1 (low) µg/ml. Pooled wells were stained with anti-CD4-CyChrome and anti-CD8-allophycocyanin, and analyzed by flow cytometry. Data are shown for live gated thymocyte subsets, using the regions shown in A. The percentages of cells that have undergone division (dilution of CFSE fluorescence) are indicated in the top left corner of each histogram. CFSE fluorescence is indicated on a log scale.

 
In anti-CD3-stimulated cultures there was an increase in the proportions of both CD4 and CD8 SP thymocytes relative to DP thymocytes as a consequence of SP proliferation and DP apoptosis in response to TCR ligation (Fig. 7A). In the cultures derived from nontransgenic mice the ratio of CD8 SP to CD4 SP cells remained constant, while this ratio increased almost 6-fold in the transgenic cultures, from 0.89 in the input thymocyte population to 4.9 after 72 h of culture with anti-CD3. Over 72 h the consequence of exposing the cells to anti-CD3 was to increase the CD8:CD4 SP ratio ~20-fold in the transgenic thymocytes vs 2-fold in the nontransgenic thymocytes.

CFSE labeling before the initiation of cultures was used to directly assess the proliferation of thymocyte subsets during culture. In the absence of anti-CD3 there was no proliferation of CD4 SP or CD8 SP thymocytes from either nontransgenic or transgenic mice (Fig. 7B). Therefore, the RasGRP1 transgene does not confer a proliferative response in the absence of TCR-mediated signaling. Using a high concentration of anti-CD3, both CD4 SP and CD8 SP thymocytes were highly proliferative, and the RasGRP1 transgene caused a moderate increase in proliferation in both subsets. When a low concentration of anti-CD3 was used in the cultures, there was very little proliferation of either CD4 SP or CD8 SP cells from nontransgenic mice. The RasGRP1 transgene moderately increased the proliferative response of CD4 SP cells, to ~0.7 divisions/cell and greatly increased the proliferative response of the CD8 SP cells, to ~3 divisions/cell. Thus, the RasGRP1 transgene had the effect of lowering the threshold of TCR signaling needed to trigger proliferation in both SP lineages, but with an augmented effect in CD8 SP thymocytes. The preferential proliferation of CD8 vs CD4 SP thymocytes in the RasGRP1 transgenic mice was particularly remarkable given the considerably lower levels of expression of CD3 on the CD8 SP thymocytes. Based on the CFSE peak areas, 40% of the input CD8 SP cells from the RasGRP1 transgenic contributed to the proliferation that occurred in response to the low dose of anti-CD3, while 90% of the input transgenic CD8 SP cells were CD3low. Therefore, the CD3low subset provided the majority of the anti-CD3-responsive CD8 SP cells in the transgenic thymocyte culture that were competent to proliferate in response to low amounts of anti-CD3.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Expression of RasGRP1 at both the mRNA and protein levels is low in most DN thymocytes, including the CD44- CD25+ DN thymocytes that are on the threshold of initiating pre-TCR signaling. Differentiation to DP thymocytes is accompanied by a large increase in RasGRP1 levels. Up-regulation of RasGRP1 expression occurs as a rapid response to the initiation of pre-TCR signaling, as demonstrated by its induction in RAG-2-/- DN thymocytes within 4 h of injection of anti-CD3. The kinetics of RasGRP1 up-regulation is equivalent to other immediate early responses to pre-TCR signaling such as CD69 expression and ERK activation. The immediate early response to pre-TCR signaling precedes by several days the appearance of differentiation markers of the DN to DP transition, e.g., CD4 and CD8 expression.

We have generated transgenic mice in which the expression of RasGRP1 is constitutively elevated in DN thymocytes to approximately the quantity attained in DP thymocytes that have undergone pre-TCR-driven differentiation. These mice have allowed us to determine the contribution of RasGRP1 to the DN to DP transition. The development of thymocytes in RAG-2-/- mice is normally arrested at the DN stage, because pre-TCR signaling is attenuated by the absence of TCR{beta}. The production of large numbers of DN and DP thymocytes in the RasGRP1 transgenic RAG-2-/- mice demonstrates that increased expression of RasGRP1 can substitute for TCR{beta} in providing signals that drive the differentiation of DN thymocytes. Our results fit a model of RasGRP1 function in which the onset of pre-TCR signaling induces RasGRP1 expression, which in turn contributes signaling capabilities that act on their own, or in conjunction with other modes of signaling provided via TCR{beta}-deficient pre-TCR, to promote proliferation and differentiation. This model predicts that RasGRP1-deficient mice should be depleted of DP thymocytes and have either normal or increased numbers of DN thymocytes depending on whether DN thymocytes that are unable to make the transition to DP accumulate or are eliminated. RasGRP1-deficient mice do have reductions and increases in DP and DN numbers, respectively, but they are relatively minor, resulting in a shift in the DP:DN ratio from 15 in the wild-type mice to 9 in the RasGRP1-deficient mice (32). Therefore, while the results from the transgenic mice indicate that RasGRP1 contributes to pre-TCR-mediated progression beyond the DN3 stage, the knockout mice clearly demonstrate that RasGRP1 is not essential for this process. There are three close relatives of RasGRP1, RasGRP2 (also known as CalDAG-GEFI) (28, 46), RasGRP3 (also known as CalDAG-GEFIII) (29, 47), and RasGRP4 (48), which are expressed in the thymus and potentially share the signal reception and transduction capabilities of RasGRP1, i.e., activation by diacylglycerol and/or calcium, and GDP to GTP exchange on Ras or Ras-related GTPases. It is possible that multiple RasGRPs (or exchange factors outside this family) are normally induced or activated via pre-TCR, with their combined signaling being sufficient to drive the DN to DP transition, but with each of them being individually surplus to the threshold for functional signaling levels.

Only 8 of 13 RasGRP transgenic RAG-2-/- mice had undergone a DN to DP transition, and among these 8 mice there was a 100-fold range of production of DP thymocytes, from 300,000 to 30 million. In contrast, mutationally activated forms of Ras or Raf can induce the emergence of high numbers of predominantly DP thymocytes in most or all RAG-deficient mice (10, 11, 12). A possible explanation for the partial penetrance of the DN to DP transition in the RasGRP1 transgenic mice is variable expression of the RasGRP1 transgene within the DN subset. Extinction of RasGRP1 transgene expression at the DN stage did occur sporadically in the 1268 line, at a frequency of ~10% (data not shown). Additionally, the reduced effectiveness of RasGRP1 relative to mutationally activated Ras or Raf in driving the DN to DP transition in RAG-deficient mice may reflect the distinct regulatory susceptibilities of these proteins. The Ras and Raf mutants are largely immune to negative regulation, are independent of positive regulation, and trigger signaling strengths well above physiological levels. In contrast, the RasGRP1 protein expressed from the transgene is in wild-type form and is expressed in DN thymocytes at levels closely equivalent to those attained during the DN to DP transition. Its susceptibility to regulation would be expected to make RasGRP1 less effective than activated Ras or Raf in complementing the deficiency in pre-TCR signaling in RAG-2-/- mice.

An unanticipated phenotype of the RasGRP1 transgenic mice was the 4-fold increase in the numbers of CD8 SP thymocytes. Atypical features of the transgenic CD8 SP population were a high proportion of CD3low cells with otherwise mature phenotypes and a reduced proportion of cells that had divided in vivo. In contrast, the CD4 SP population was at normal numbers and had normal levels of CD3 and other markers. The atypical CD8 SP thymocytes did not appear in RAG-2-/- mice and were eliminated in male mice expressing the H-Y TCR (A. M. Norment et al., unpublished observations), indicating that their development was dependent on TCR and was subject to negative selection.

The RasGRP1 transgene showed a strong selectivity for the CD8 SP lineage in its ability to enhance responses to CD3 cross-linking. While the RasGRP1 transgene conferred proliferation to suboptimal levels of anti-CD3 in both lineages, there was a much more pronounced effect in the CD8 SP thymocytes in terms of both the proportion of cells that entered the cell cycle and the average number of divisions that they achieved. The preferential effect of the RasGRP1 transgene on CD8 SP thymocytes occurred despite the fact that most CD8 SP thymocytes in the transgenic mice had atypically low levels of CD3, while the CD4 SP thymocytes had normal levels of CD3. At higher anti-CD3 concentrations, the proliferation-enhancing effect of the RasGRP1 transgene was less evident, particularly in terms of its ability to selectively promote division among CD8 SP thymocytes. Despite this, 3-day culture in high anti-CD3 caused a >10-fold increase in the CD8 to CD4 ratio among transgenic vs nontransgenic thymocytes. These effects on the responses of cultured thymocytes to TCR cross-linking suggest that RasGRP1 may promote the maintenance or expansion of thymocytes after they have entered the CD8 lineage, while failing to have an equivalent effect within the CD4 lineage.

The emergence of CD8 SP thymocytes with atypically low levels of CD3 raises the possibility that TCR selection is being perturbed by the RasGRP1 transgene in a way that augments the output of CD8 SP cells while having no effect on the output of CD4 SP cells. There is considerable evidence that strong signaling from TCR/coreceptor through Lck to ERK is required for DP thymocytes to become committed to and/or survive within the CD4 SP lineage, with CD8 commitment or maintenance being achieved only when signaling is weaker, but still sufficient to achieve positive selection (4, 49, 50). Augmentation of weak CD8-coupled Lck signaling by RasGRP1 overexpression may move MHC class I-restricted CD3low thymocytes into a signaling strength range that would be sufficient for survival via positive selection. In contrast, among MHC class II-restricted thymocytes, the effect on signal strength provided by increased RasGRP1 may be inconsequential relative to the strong signaling provided by CD4-coupled Lck. Positive selection occurs predominantly within the DP stage, and therefore it would be expected that if the RasGRP1 transgene is perturbing positive selection it would probably do so at the DP stage. However, the 1198 and 1268 lines were equivalent in their overproduction of CD3low CD8 SP cells and had high transgene expression in most CD8 SP cells, but only the 1268 line had obvious transgene expression in the majority of DP cells. Therefore, if the RasGRP1 transgene is causing overproduction of CD3low CD8 SP thymocytes by affecting selection at the DP stage, then it must have a low expression threshold for doing so or must act on a minor population of DP cells. An alternative explanation is that RasGRP1 contributes to ongoing positive selection of CD3low thymocytes after they have lost CD4 expression.

RasGRP1 may augment CD8 SP production by signaling pathways that are not initiated by H-Ras and do not flow through to the ERK kinases. There are discrepancies in the SP thymocyte phenotypes of RasGRP1 transgenic vs other transgenic or knockout mice that suggest this may be true. Grb2 hemizygous mice have normal numbers of CD4 and CD8 cells despite having greatly reduced Ras activation via TCR (26). Mice with thymocyte-specific transgenic expression of constitutively activated H-Ras also have normal numbers of CD4 SP and CD8 SP thymocytes and normal expression levels of TCR (11). The expression of dominant negative H-Ras equally reduces numbers of CD8 SP and CD4 SP thymocytes (25). The expression of membrane-localized Raf-1 (12) causes a 2-fold increase in the number of CD8 SP thymocytes, but in contrast to the RasGRP1 transgenics, this is accompanied by a 2-fold reduction in the number of CD4 SP thymocytes. RasGRP1 has the potential to activate a variety of Ras family members that are functionally diverse in terms of signaling localization and effector specificity. It will be a considerable challenge to determine how RasGRP1 and its three relatives act in concert to influence thymocyte development through the selective activation of multiple Ras family members.


   Acknowledgments
 
We gratefully acknowledge K. A. Forbush for generation of RasGRP1 transgenic mice. We thank K. Allen for flow cytometric analysis, Xiao Cun Pan and Carolyn Bateman for mouse care, and E. M. Jacobson for PCR screening of the mice. We also thank Steve Levin for critical reading of this manuscript, as well as J. Yamagiwa for secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the National Cancer Institute (CA64448) and the Howard Hughes Medical Institute (to M.J.B.) and from the Canadian Institutes for Health Research (to R.J.K.). Back

2 Current address: Amgen Corp., 51 University Street, Seattle, WA 98101. Back

3 Current address: Corixa Corp., 1124 Columbia, Suite 200, Seattle, WA 98104. Back

4 Current address: Primal, Inc., Suite 650, 1124 Columbia Street, Seattle, WA 98104. Back

5 Address correspondence and reprint requests to Dr. Robert J. Kay, Terry Fox Laboratory, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 4E6. E-mail: robert{at}terryfox.ubc.ca Back

6 Abbreviations used in this paper: DN, double negative; BrdU, 5-bromodeoxyuridine; DP, double positive; HA, hemagglutinin; HPRT, hypoxanthine phosphoribosyltransferase; MEK, mitogen-activated protein kinase kinase; RAG, recombinase-activating gene; SP, single positive; Tg, transgene or transgenic. Back

Received for publication May 3, 2002. Accepted for publication November 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kruisbeek, A. M., M. C. Haks, M. Carleton, A. M. Michie, J. C. Zuniga-Pflucker, D. L. Wiest. 2000. Branching out to gain control: how the pre-TCR is linked to multiple functions. Immunol. Today 21:637.[Medline]
  2. von Boehmer, H., I. Aifantis, O. Azogui, J. Feinberg, C. Saint-Ruf, C. Zober, C. Garcia, J. Buer. 1998. Crucial function of the pre-T-cell receptor (TCR) in TCR{beta} selection, TCR{beta} allelic exclusion and {alpha}{beta} versus {gamma}{delta} lineage commitment. Immunol. Rev. 165:111.[Medline]
  3. von Boehmer, H., H. J. Fehling. 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15:433.[Medline]
  4. Hogquist, K. A.. 2001. Signal strength in thymic selection and lineage commitment. Curr. Opin. Immunol. 13:225.[Medline]
  5. Rincon, M.. 2001. MAP-kinase signaling pathways in T cells. Curr. Opin. Immunol. 13:339.[Medline]
  6. Genot, E., D. A. Cantrell. 2000. Ras regulation and function in lymphocytes. Curr. Opin. Immunol. 12:289.[Medline]
  7. Berg, L. J., J. Kang. 2001. Molecular determinants of TCR expression and selection. Curr. Opin. Immunol. 13:232.[Medline]
  8. Michie, A. M., S. Trop, D. L. Wiest, J. C. Zuniga-Pflucker. 1999. Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo. J. Exp. Med. 190:1647.[Abstract/Free Full Text]
  9. Crompton, T., K. C. Gilmour, M. J. Owen. 1996. The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte. Cell 86:243.[Medline]
  10. Swat, W., Y. Shinkai, H. L. Cheng, L. Davidson, F. W. Alt. 1996. Activated Ras signals differentiation and expansion of CD4+8+ thymocytes. Proc. Natl. Acad. Sci. USA 93:4683.[Abstract/Free Full Text]
  11. Gartner, F., F. W. Alt, R. Monroe, M. Chu, B. P. Sleckman, L. Davidson, W. Swat. 1999. Immature thymocytes employ distinct signaling pathways for allelic exclusion versus differentiation and expansion. Immunity 10:537.[Medline]
  12. Iritani, B. M., J. Alberola-Ila, K. A. Forbush, R. M. Perimutter. 1999. Distinct signals mediate maturation and allelic exclusion in lymphocyte progenitors. Immunity 10:713.[Medline]
  13. Alberola-Ila, J., K. A. Hogquist, K. A. Swan, M. J. Bevan, R. M. Perlmutter. 1996. Positive and negative selection invoke distinct signaling pathways. J. Exp. Med. 184:9.[Abstract/Free Full Text]
  14. Alberola-Ila, J., K. A. Forbush, R. Seger, E. G. Krebs, R. M. Perlmutter. 1995. Selective requirement for MAP kinase activation in thymocyte differentiation. Nature 373:620.[Medline]
  15. Sugawara, T., T. Moriguchi, E. Nishida, Y. Takahama. 1998. Differential roles of ERK and p38 MAP kinase pathways in positive and negative selection of T lymphocytes. Immunity 9:565.[Medline]
  16. Delgado, P., E. Fernandez, V. Dave, D. Kappes, B. Alarcon. 2000. CD3{delta} couples T-cell receptor signalling to ERK activation and thymocyte positive selection. Nature 406:426.[Medline]
  17. Werlen, G., B. Hausmann, E. Palmer. 2000. A motif in the {alpha}{beta} T-cell receptor controls positive selection by modulating ERK activity. Nature 406:422.[Medline]
  18. O’Shea, C. C., T. Crompton, I. R. Rosewell, A. C. Hayday, M. J. Owen. 1996. Raf regulates positive selection. Eur. J. Immunol. 26:2350.[Medline]
  19. Mariathasan, S., S. S. Ho, A. Zakarian, P. S. Ohashi. 2000. Degree of ERK activation influences both positive and negative thymocyte selection. Eur. J. Immunol. 30:1060.[Medline]
  20. Bommhardt, U., Y. Scheuring, C. Bickel, R. Zamoyska, T. Hunig. 2000. MEK activity regulates negative selection of immature CD4+CD8+ thymocytes. J. Immunol. 164:2326.[Abstract/Free Full Text]
  21. Sharp, L. L., S. M. Hedrick. 1999. Commitment to the CD4 lineage mediated by extracellular signal-related kinase mitogen-activated protein kinase and lck signaling. J. Immunol. 163:6598.[Abstract/Free Full Text]
  22. Sharp, L. L., D. A. Schwarz, C. M. Bott, C. J. Marshall, S. M. Hedrick. 1997. The influence of the MAPK pathway on T cell lineage commitment. Immunity 7:609.[Medline]
  23. Bommhardt, U., M. A. Basson, U. Krummrei, R. Zamoyska. 1999. Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus. J. Immunol. 163:715.[Abstract/Free Full Text]
  24. Feig, L. A.. 1999. Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases. Nat. Cell Biol. 1:E25.[Medline]
  25. Swan, K. A., J. Alberola-Ila, J. A. Gross, M. W. Appleby, K. A. Forbush, J. F. Thomas, R. M. Perlmutter. 1995. Involvement of p21ras distinguishes positive and negative selection in thymocytes. EMBO J. 14:276.[Medline]
  26. Gong, Q., A. M. Cheng, A. M. Akk, J. Alberola-Ila, G. Gong, T. Pawson, A. C. Chan. 2001. Disruption of T cell signaling networks and development by Grb2 haploid insufficiency. Nat. Immunol. 2:29.[Medline]
  27. Ebinu, J. O., D. A. Bottorff, E. Y. Chan, S. L. Stang, R. J. Dunn, J. C. Stone. 1998. RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Science 280:1082.[Abstract/Free Full Text]
  28. Kawasaki, H., G. M. Springett, S. Toki, J. J. Canales, P. Harlan, J. P. Blumenstiel, E. J. Chen, I. A. Bany, N. Mochizuki, A. Ashbacher, et al 1998. A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia [published erratum appears in Proc. Natl. Acad. Sci. USA 1999 Jan 5;96(1):318]. Proc. Natl. Acad. Sci. USA 95:13278.[Abstract/Free Full Text]
  29. Ohba, Y., N. Mochizuki, S. Yamashita, A. M. Chan, J. W. Schrader, S. Hattori, K. Nagashima, M. Matsuda. 2000. Regulatory proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3. J. Biol. Chem. 275:20020.[Abstract/Free Full Text]
  30. Tognon, C. E., H. E. Kirk, L. A. Passmore, I. P. Whitehead, C. J. Der, R. J. Kay. 1998. Regulation of RasGRP via a phorbol ester-responsive C1 domain. Mol. Cell. Biol. 18:6995.[Abstract/Free Full Text]
  31. Ebinu, J. O., S. L. Stang, C. Teixeira, D. A. Bottorff, J. Hooton, P. M. Blumberg, M. Barry, R. C. Bleakley, H. L. Ostergaard, J. C. Stone. 2000. RasGRP links T-cell receptor signaling to Ras. Blood 95:3199.[Abstract/Free Full Text]
  32. Dower, N. A., S. L. Stang, D. A. Botorff, J. O. Ebinu, P. Dickie, H. L. Ostergaard, J. C. Stone. 2000. RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat. Immunol. 1:317.[Medline]
  33. Norment, A. M., L. Y. Bogatzki, B. N. Gantner, M. J. Bevan. 2000. Murine CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regulated following pre-TCR signaling. J. Immunol. 164:639.[Abstract/Free Full Text]
  34. Goldrath, A. W., L. Y. Bogatzki, M. J. Bevan. 2000. Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation. J. Exp. Med. 192:557.[Abstract/Free Full Text]
  35. Chaffin, K. E., C. R. Beals, T. M. Wilkie, K. A. Forbush, M. I. Simon, R. M. Perlmutter. 1990. Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase. EMBO J. 9:3821.[Medline]
  36. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall. 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855.[Medline]
  37. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68:869.[Medline]
  38. Levelt, C. N., P. Mombaerts, B. Wang, H. Kohler, S. Tonegawa, K. Eichmann, C. Terhorst. 1995. Regulation of thymocyte development through CD3: functional dissociation between p56lck and CD3{varsigma} in early thymic selection. Immunity 3:215.[Medline]
  39. Levelt, C. N., P. Mombaerts, A. Iglesias, S. Tonegawa, K. Eichmann. 1993. Restoration of early thymocyte differentiation in T-cell receptor {beta}-chain-deficient mutant mice by transmembrane signaling through CD3{epsilon}. Proc. Natl. Acad. Sci. USA 90:11401.[Abstract/Free Full Text]
  40. Nagy, E., L. E. Maquat. 1998. A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends Biochem. Sci. 23:198.[Medline]
  41. Anderson, S. J., C. Coleclough. 1993. Regulation of CD4 and CD8 expression on mouse T cells: active removal from the cell surface by two mechanisms. J. Immunol. 151:5123.[Abstract]
  42. Azzam, H. S., A. Grinberg, K. Lui, H. Shen, E. W. Shores, P. E. Love. 1998. CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity. J. Exp. Med. 188:2301.[Abstract/Free Full Text]
  43. D’Ambrosio, D., D. A. Cantrell, L. Frati, A. Santoni, R. Testi. 1994. Involvement of p21ras activation in T cell CD69 expression. Eur. J. Immunol. 24:616.[Medline]
  44. Gabor, M. J., D. I. Godfrey, R. Scollay. 1997. Recent thymic emigrants are distinct from most medullary thymocytes. Eur. J. Immunol. 27:2010.[Medline]
  45. Anderson, G., K. J. Hare, E. J. Jenkinson. 1999. Positive selection of thymocytes: the long and winding road. Immunol. Today 20:463.[Medline]
  46. Clyde-Smith, J., G. Silins, M. Gartside, S. Grimmond, M. Etheridge, A. Apolloni, N. Hayward, J. F. Hancock. 2000. Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor. J. Biol. Chem. 275:32260.[Abstract/Free Full Text]
  47. Lorenzo, P. S., J. W. Kung, D. A. Bottorff, S. H. Garfield, J. C. Stone, P. M. Blumberg. 2001. Phorbol esters modulate the Ras exchange factor RasGRP3. Cancer Res. 61:943.[Abstract/Free Full Text]
  48. Yang, Y., L. Li, G. W. Wong, S. A. Krilis, M. S. Madhusudhan, A. Sali, R. L. Stevens. 2002. RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function. J. Biol. Chem. 277:25756.[Abstract/Free Full Text]
  49. Legname, G., B. Seddon, M. Lovatt, P. Tomlinson, N. Sarner, M. Tolaini, K. Williams, T. Norton, D. Kioussis, R. Zamoyska. 2000. Inducible expression of a p56Lck transgene reveals a central role for Lck in the differentiation of CD4 SP thymocytes. Immunity 12:537.[Medline]
  50. Salmon, P., M. Mong, X. J. Kang, D. Cado, E. Robey. 1999. The role of CD8{alpha}' in the CD4 versus CD8 lineage choice. J. Immunol. 163:5312.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Yasuda, R. L. Stevens, T. Terada, M. Takeda, T. Hashimoto, J. Fukae, T. Horita, H. Kataoka, T. Atsumi, and T. Koike
Defective Expression of Ras Guanyl Nucleotide-Releasing Protein 1 in a Subset of Patients with Systemic Lupus Erythematosus
J. Immunol., October 1, 2007; 179(7): 4890 - 4900.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
N. Beaulieu, B. Zahedi, R. E. Goulding, G. Tazmini, K. V. Anthony, S. L. Omeis, D. R. de Jong, and R. J. Kay
Regulation of RasGRP1 by B Cell Antigen Receptor Requires Cooperativity between Three Domains Controlling Translocation to the Plasma Membrane
Mol. Biol. Cell, August 1, 2007; 18(8): 3156 - 3168.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
P. Trampont, L. Zhang, and K. S. Ravichandran
ShcA Mediates the Dominant Pathway to Extracellular Signal-Regulated Kinase Activation during Early Thymic Development
Mol. Cell. Biol., December 1, 2006; 26(23): 9035 - 9044.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
X. Yu, K. Bauer, P. Wernhoff, D. Koczan, S. Moller, H.-J. Thiesen, and S. M. Ibrahim
Fine Mapping of Collagen-Induced Arthritis Quantitative Trait Loci in an Advanced Intercross Line
J. Immunol., November 15, 2006; 177(10): 7042 - 7049.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. Guilbault and R. J. Kay
RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis
J. Biol. Chem., May 7, 2004; 279(19): 19523 - 19530.
[Abstract] [Full Text] [PDF]