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The Journal of Immunology, 2006, 176: 6824-6830.
Copyright © 2006 by The American Association of Immunologists

A Role for MAPK in Feedback Inhibition of Tcrb Recombination1

Annette M. Jackson and Michael S. Krangel2

Department of Immunology, Duke University Medical Center, Durham NC 27710


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4–CD8– double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4+CD8+ double-positive (DP) differentiation, proliferation, and feedback inhibition of further Vbeta to DJbeta rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of Vbeta gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of Vbeta chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vbeta chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vbeta segment accessibility and normal feedback inhibition of the Vbeta to DJbeta rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell receptor diversity is achieved by a unique process of somatic recombination that directs the combinatorial joining of variable (V), diversity (D), and joining (J) gene segments during T cell development (1, 2). V(D)J recombination at TCR loci depends on the products of the RAG genes (RAG proteins) which bind and cleave the recombination signal sequences (RSSs)3 flanking each V, D, and J gene segment. Recombination at the various loci is regulated according to cell lineage and developmental stage. Moreover, at the Tcrb locus, recombination is constrained by allelic exclusion mechanisms that restrict each cell to the production of a single functional VDJbeta rearrangement (3).

Tcrb recombination occurs in DN thymocytes, beginning with Dbeta to Jbeta rearrangement on both alleles followed by Vbeta to DJbeta rearrangement on a single allele. If a functional TCRbeta protein is generated, it assembles with an invariant pre-T{alpha} protein and CD3 proteins (CD3{delta}, {epsilon}, {gamma}, and {zeta} subunits) to form a pre-TCR (4, 5). Signaling via the pre-TCR results in the transient down-regulation of RAG, entry into cell cycle, and differentiation to the DP compartment. In addition, pre-TCR signaling initiates feedback inhibition of further VDJbeta recombination, which is essential for allelic exclusion. Thymocytes that do not express a functional TCRbeta protein do not expand or progress to the DP stage as a result of a checkpoint known as "beta selection" (6). Successful transition to the DP compartment allows for a second wave of Rag expression and the initiation of Tcra recombination.

Allelic exclusion of the Tcrb locus is associated with an inactivation of the Vbeta gene segments at the DN to DP transition. This loss in Vbeta segment activation is measured by decreased germline transcription, decreased histone H3 and H4 acetylation, decreased histone H3 lysine 4 (K4) methylation, and decreased sensitivity to nuclease digestion (7, 8, 9). This loss in Vbeta accessibility no doubt contributes to feedback inhibition and allelic exclusion. However, recent studies indicate that these processes are multifactorial, with reduced accessibility of the Vbeta gene segments representing only one component of the inhibitory program (9, 10).

The critical downstream proteins involved in pre-TCR signaling have been mapped through gene targeting studies and the use of constitutively active or dominant-negative transgenes. Similar to TCR signaling, the pre-TCR immunoreceptor tyrosine-based activation motifs are phosphorylated by Src family kinases such as Lck (11, 12). Once phosphorylated, these motifs become docking sites for the Syk family kinase Zap70 (13). Adapter proteins such as the linker for activation of T cells (LAT) and the Src homology 2 domain-containing leukocyte protein (SLP-76) couple these active kinases with the downstream effector proteins PLC{gamma}1, and Ras (14, 15, 16, 17, 18). Activation of PLC{gamma}1 results in the mobilization of intracellular calcium and the activation of protein kinase C (PKC) (19). Ras is critical for the activation of the Raf-MAPK-ERK signaling pathway (20, 21). Defects in the pre-TCR components or signaling proteins result in reduced thymic cellularity, reduced DN to DP differentiation, and a loss of allelic exclusion (4, 5).

The current view of pre-TCR signaling suggests branch points in the cascade that impact different biological outcomes. The expression of a constitutively active Lck (LckF505), Prkca (PKC{alpha}-CAT), Hras1 (Rasv12), or Raf1 (Raf-CAAX) transgene has been demonstrated to promote DP differentiation in pre-TCR-deficient mice (11, 19, 22, 23). In addition, the LckF505 and PKC{alpha}-CAT transgenes provide allelic exclusion signals as shown by the inhibition of Vbeta to DJbeta rearrangements at the endogenous Tcrb locus (11, 19). However, developing thymocytes containing Rasv12 or Raf-CAAX transgenes displayed normal levels of endogenous Vbeta to DJbeta rearrangement (22, 23). Thus, Rasv12 and Raf-CAAX are thought to promote DN to DP differentiation but not contribute to allelic exclusion. Because deletion of Lcp2 (encoding SLP-76) results in a loss of thymocyte differentiation and a loss of allelic exclusion (24), it was concluded that the signaling pathways involved in differentiation and allelic exclusion diverge downstream of SLP-76 but upstream of Ras (4, 5).

To better understand the relationship between pre-TCR signaling and the developmental changes at the Tcrb locus associated with allelic exclusion, we analyzed the properties of DP thymocytes generated by a constitutively active Raf-CAAX transgene. Consistent with previous publications, we show that the Raf-CAAX transgene does not extinguish Tcrb recombination in DN thymocytes. However, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vbeta segment transcription and accessibility and normal feedback inhibition of Vbeta to DJbeta rearrangement. Thus, although the MAPK pathway is not sufficient to enforce Tcrb allelic exclusion, it clearly contributes to the feedback inhibited state in DP thymocytes. Our data suggest distinct requirements for Tcrb feedback inhibition in DN and DP thymocytes and indicate that a component of the feedback mechanism is tightly linked to DP differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6 (B6), 129, Rag2–/– mice, Rag2–/– x Tcrb transgenic mice (Rxbeta) (25), Raf-CAAX transgenic mice (23), and Lat–/– mice (17) were housed at the Duke University Vivarium. Lat–/– mice were a gift from W. Zhang (Duke University, Durham NC). Raf-CAAX transgenic mice were a gift from R. Perlmutter (Amgen, Thousand Oaks, CA). All mice were used in accordance with protocols approved by the Duke University Institutional Animal Care and Use Committee.

Flow cytometric analysis and thymocyte isolation

Surface staining was performed on thymocytes according to standard staining protocols. All Abs were purchased from BD Pharmingen. In all instances, cells were initially incubated for 5 min with a mAb specific for CD16 and CD32 (clone 2.4G2) to block nonspecific staining. Analysis and sorting of DN and DP thymocytes was conducted using FITC-conjugated anti-CD4 (clone GK1.5) and PE-conjugated anti-CD8 (clone 53-6.7). The exclusion dye 7-aminoactinomycin D was included in all cell-sorting experiments. To perform intracellular TCRbeta staining, thymocytes were surface stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8. The thymocytes were then permeabilized and stained intracellularly with CyChrome-conjugated anti-TCRbeta (clone H57-597) according to the Cytoperm/Cytofix kit (BD Pharmingen). TCRbeta staining was evaluated in the CD4+CD8+ population. Cell sorting and analysis were conducted using a FACStarPlus (BD Biosciences) or FACSVantage SE (BD Biosciences) and CellQuest software.

RT-PCR

RNA was extracted from cells using TRIzol (Invitrogen Life Technologies) according to the manufacturer’s instructions. For germline transcription analysis, contaminating genomic DNA was removed with DNA-free (Ambion) according to the manufacturer’s instructions, and cDNA synthesis was performed with Transcriptor (Roche) and random hexamer primers (Roche) according to the Roche protocol. For analysis of Rag1 and Rag2 expression, cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) and an oligo(dT) primer (Invitrogen Life Technologies) according to manufacturer’s protocol. PCR analysis of germline transcription was performed on 3-fold serial dilutions of cDNA using a "touchdown" PCR strategy: 5 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at annealing temperature, and 1 min at 72°C, and a 10-min extension at 72°C. Annealing temperature was held at 68, 65, and 62°C for 5 cycles each and at 58°C for 17 cycles. Amplicons were electrophoresed through agarose gels and visualized by ethidium bromide staining. Vbeta primers used for germline transcription were positioned in the leader sequence and downstream of the RSS, respectively. Primers for Vbeta11, Vbeta12, Dbeta1, and Act-b (9) and Tcrg (26) were previously described. Tcra primers were: 5'-AGAACCTGCTGTGTACCAGTTA-3' and 5'-GAGTCAGGCTCTGTCAGTCTT-3'.

Chromatin immunoprecipitation

Thymocytes were harvested and mononucleosomes were prepared as previously described (27). Mononucleosomes (25 µg) were immunoprecipitated with 5 µg each of Abs against diacetylated histone H3, tetraacetylated histone H4, dimethylated histone H3K4, and control rabbit IgG (Upstate Biotechnology). The bound and input fractions were quantified by real-time PCR using SYBR green and a Light Cycler (Roche). Ratios of bound:input were calculated and were normalized to those for carbamoyltransferase dihydrorotase (Cad) in each sample. Primers for Vbeta12, Vbeta13, T4/T5, and Cad have been previously described (9). Dbeta1 primers were: 5'-GATCCAGAATGCTTTCACG-3' and 5'-CTGCATCCTTTGCTGCTA-3'.

Analysis of V(D)J recombination products

VDJbeta and DJbeta coding joints were analyzed by touchdown PCR (as described above) using as a template genomic DNA prepared from thymocytes. To analyze signal end intermediates, thymocyte genomic DNA was extracted and linker ligation was performed as previously described (28). Linker-ligated DNA was then used to amplify signal end intermediates by touchdown PCR (as described above). Cd14 was amplified by PCR as follows: 94°C for 5 min, followed by 20 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 1 min, and a 10-min extension at 72°C. Amplicons were electrophoresed through agarose gels and analyzed by Southern blot using 32P-labeled oligonucleotide probes. The signal end primer for Vbeta6 was: 5'-GTCGGTAGCTACTTTGTTTCAAGTCC-3'. Primers and probes for Vbeta11, Vbeta12, Vbeta13, Dbeta2, Jbeta2, and Cd14 (9) and the linker and linker primer (29) were described previously. For sequence analysis, amplified Vbeta12 coding joints were purified through agarose gels, cloned using a TOPO TA Cloning kit for Sequencing (Invitrogen Life Technologies), and sequenced using a model 3730 DNA Analyzer (Applied Biosystems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Analysis of gene expression and chromatin structure in Raf-CAAX-derived DP thymocytes

To analyze the properties of DP thymocytes generated by MAPK activation, we bred the Raf-CAAX transgene onto a Rag2–/– background (RxRC) and examined the expression of various genes. Expression of the Raf-CAAX transgene is driven by the lck proximal promoter and IgH chain enhancer and is primarily restricted to immature lymphocytes (23). This Raf protein is constitutively active due to a farnesylation signal (CAAX) which provides targeting to the cell membrane, mimicking normal Ras activation. Consistent with a previous publication (23), Raf-CAAX signals generated DP thymocytes with normal CD4 and CD8 expression. However, we observed a delay in thymocyte differentiation (Fig. 1A) and variable thymic cellularity between RxRC littermates (10–40 x 106 in 4- to 7-wk-old mice). RT-PCR was performed on purified RxRC DP thymocytes as well as DN (Rag2–/–) and DP (Rag2–/– x Tcrb transgene, Rxbeta) thymocyte control populations. This analysis showed that RxRC DP thymocytes were similar to Rxbeta with respect to the level of Tcrg, Tcra, Vbeta11, and Vbeta12 germline transcription (Fig. 1B). The only discrepancy was that RxRC DP thymocytes displayed reduced germline Dbeta1 transcripts as compared with the DP (Rxbeta) control. The significance of this observation is unknown.


Figure 1
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  		FIGURE 1. DP differentiation induced by a constitutively active Raf-CAAX transgene in Rag2–/– thymocytes. A, CD4 and CD8 expression in 4- and 7-wk-old RxRC mice was analyzed using flow cytometry. B, RT-PCR analysis of gene expression patterns in DN thymocytes (Rag2–/–), DP thymocytes (Rxbeta), and purified DP thymocytes from RxRC mice (80 and 94% purity). PCR was performed on 3-fold serial dilutions of cDNA (indicated by wedges) and on control samples prepared without reverse transcriptase (–). A sample without DNA (H2O) served as a negative control and RT-PCR of Act-b was used to assess cDNA loading.

 
Thymocyte differentiation from DN to DP is also accompanied by a loss in histone H3 and H4 acetylation and histone H3K4 dimethylation within germline Vbeta gene segments. Chromatin immunoprecipitation was performed using Abs specific for diacetylated histone H3, tetraacetylated histone H4, and dimethylated histone H3K4 in DN (Rag2–/–), DP (Rxbeta), and unfractionated RxRC thymocytes (69 and 77% DP). Immunoprecipitated chromatin was analyzed by real-time PCR at various sites within the Tcrb locus. Levels of histone acetylation and H3K4 dimethylation were high at the promoters (P) and RSSs of Vbeta gene segments in the DN control but were decreased in both Raf-CAAX and control Rxbeta DP thymocytes. Nevertheless, all samples analyzed displayed equivalent levels of histone H3 and H4 acetylation and histone H3K4 dimethylation at control sites Dbeta1 and trypsinogen (T4/T5) (Fig. 2). Thus, DP thymocytes generated by the Raf-CAAX transgene display gene expression patterns and Vbeta chromatin changes similar to those seen in DP thymocytes generated by a complete pre-TCR signaling cascade.


Figure 2
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  		FIGURE 2. Tcrb locus chromatin structure in thymocytes of RxRC mice. Chromatin immunoprecipitation was performed on mononucleosomes prepared from DN thymocytes (Rag2–/–), DP thymocytes (Rxbeta), and unfractionated thymocytes from RxRC mice (69 and 77% DP). Immunoprecipitations were analyzed at Vbeta promoter (P) and RSS sites, just downstream of Dbeta1, and between inactive trypsinogen genes (T4/T5) located upstream of the Vbeta cluster. Bound and input fractions were quantified using real-time PCR and ratios of bound:input were expressed relative to the values for Cad in each sample. The data shown are the mean ± SEM for triplicate PCRs from two independent mononucleosome preparations.

 
LAT is required for allelic exclusion

We sought to analyze the potential for ongoing Tcrb locus recombination in DP thymocytes generated solely by a Raf signal. Expression of the Raf-CAAX transgene on a wild-type background would not allow us to distinguish between DP thymocytes arising from Raf signals and those arising from a full complement of pre-TCR signals. For this reason, we attempted to eliminate pre-TCR signaling potential upstream of Ras in recombinase-sufficient thymocytes. We predicted that if we bred Raf-CAAX onto a Lat–/– background (LxRC), the resulting DP thymocytes would be generated by Raf-CAAX signals alone. LAT is a proximal pre-TCR signaling protein required for normal thymocyte development (17). Thymocytes deficient in LAT are completely blocked at the DN to DP transition, despite efficient VDJbeta recombination. To determine whether this adaptor molecule is essential for propagating allelic exclusion signals, we bred a rearranged Tcrb transgene onto a Lat–/– background and analyzed the endogenous Vbeta to DJbeta rearrangement. Predictably, the Tcrb transgene did not rescue the block in DN to DP differentiation in Lat–/– thymocytes (Fig. 3A). Southern blot analysis of PCR-amplified coding joints revealed Dbeta to Jbeta rearrangement irrespective of LAT or Tcrb transgene expression (Fig. 3B). However, whereas Vbeta to DJbeta rearrangement was dramatically inhibited in Lat+/– thymocytes containing a Tcrb transgene, the Tcrb transgene had no apparent effect on Vbeta to DJbeta rearrangement in Lat–/– thymocytes. Therefore, we conclude that the adaptor protein LAT is required to efficiently propagate feedback inhibition signals emanating from the pre-TCR.


Figure 3
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  		FIGURE 3. Defective thymocyte differentiation and Tcrb allelic exclusion in LAT-deficient mice. A, Flow cytometric analysis of CD4 and CD8 expression in Lat–/– thymocytes in the absence or presence of a Tcrb transgene. B, PCR analysis of Dbeta2Jbeta2 and Vbeta13DJbeta2 coding joints in Lat+/– and Lat–/– thymocytes in the presence (+) or absence (–) of a Tcrb transgene. Three-fold serial dilutions (wedges) of genomic DNA were amplified and analyzed by Southern blot. A sample without DNA (–) served as negative control, and Cd14 amplification was used to assess DNA loading. The data are representative of two independent experiments.

 
Feedback inhibition in Raf-CAAX-derived DP thymocytes

We next examined recombinase activity at the Tcrb locus in LxRC DN and DP thymocytes by using ligation-mediated PCR to detect signal end intermediates indicative of ongoing recombination. To serve as control samples, genomic DNA was extracted from purified DP thymocytes from Lat+/– mice in the presence or absence of the Raf-CAAX transgene. Signal end intermediates were easily detected at Vbeta6 and Vbeta11 in LxRC DN thymocytes but were dramatically reduced in all DP samples (Fig. 4A). One explanation for a lack of signal end intermediates would be defective Rag expression in LxRC DP thymocytes. To address this, we isolated RNA from purified LxRC DN and DP thymocytes and performed RT-PCR for Rag1 (Fig. 4B) and Rag2 (data not shown). Expression of Rag1 and Rag2 in LxRC DN and DP thymocytes was comparable to the Lat+/– DP control samples. A second explanation would be a lack of available DJbeta substrate, due to high level Vbeta to DJbeta recombination in the DN compartment. However, PCR analysis of genomic DNA from unfractionated (Fig. 4C) or DP fractionated (Fig. 4D) LxRC thymocytes revealed levels of DJbeta2 recombination substrates that were equivalent to the Lat+/– controls. Thus, the Raf-CAAX transgene fails to suppress Vbeta to DJbeta rearrangement on either a Lat+/– or Lat–/– background as measured in total thymocytes or fractionated DN thymocytes (Fig. 4, A and C). However, DP thymocytes generated by the Raf-CAAX transgene display normal inhibition of Tcrb locus recombination, despite ongoing Rag expression and available DJbeta2 substrates.


Figure 4
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  		FIGURE 4. Feedback inhibition of Tcrb recombination in LxRC DP thymocytes. A, Detection of signal end recombination intermediates in Lat+/– DP thymocytes (95% purity), Lat+/– DP thymocytes containing the Raf-CAAX transgene (97% purity), or in DN and DP thymocytes from LxRC mice (86–97% purity). The presence (+) or absence (–) of the Raf-CAAX transgene (RC) is indicated. Linker-ligated DNA from splenocytes (S), non-linker-ligated DNA from thymocytes (T), and a sample without DNA (–) were analyzed as negative controls. Cd14 amplification was used to assess DNA loading. The data are representative of two independent experiments. B, RT-PCR analysis of Rag1 expression in DN and DP thymocytes from Lat+/– DP thymocytes (96% purity), in DN and DP thymocytes from LxRC mice (87–92% purity), and in Lat+/– DP thymocytes containing the Raf-CAAX transgene (94% purity). Two-fold serial dilutions of cDNA (wedges) and a sample without cDNA (–) were amplified and analyzed by ethidium bromide staining. C, PCR analysis of DJbeta2 and Vbeta13DJbeta2 coding joints in unfractionated thymocytes from Lat+/– and Lat–/– mice in the presence or absence of the Raf-CAAX transgene (RC). Three-fold serial dilutions of genomic DNA (wedges) were amplified with primers upstream of the Dbeta2 or Vbeta13 gene segment and downstream of the Jbeta2.7 gene segment. DNA isolated from kidney (K) and a sample without DNA (–) served as negative controls. Cd14 amplification was used to assess DNA loading. The data are representative of three independent experiments. D, PCR analysis of DJbeta2 coding joints in fractionated DN and DP thymocytes from Lat+/– and Lat–/– mice in the presence or absence of the Raf-CAAX transgene (RC). Three-fold serial dilutions of genomic DNA (wedges) were amplified with primers upstream of Dbeta2 and downstream of Jbeta2.7. DNA isolated from kidney (K) and a sample without DNA (–) served as negative controls. Cd14 amplification was used to assess DNA loading. The data are representative of three independent experiments.

 
Enforcement of beta selection in Lat–/–Raf-CAAX thymocytes

Because MAPK activation can induce DP differentiation on a Rag2–/– background (Fig. 1A), it would be expected that the DN to DP transition in LxRC thymocytes could occur in the absence of TCRbeta expression and as a consequence without beta selection. Therefore, we evaluated the presence of functional VDJbeta rearrangements in DP thymocytes using intracellular TCRbeta staining. Under the constraints of beta selection, all DP thymocytes must express a functional TCRbeta chain. If these constraints are removed, only 55%, reflecting the probability of an in-frame rearrangement on either of the two alleles, would be expected to do so. Surprisingly, essentially all DP thymocytes in LxRC mice expressed a functional TCRbeta protein, suggesting that beta selection was intact (Fig. 5A). To verify this result, genomic DNA was isolated from purified Lat+/+ and LxRC DP thymocytes and Vbeta12Dbeta2Jbeta2 coding joints were amplified by PCR. Assuming no allelic exclusion and no beta selection, only 33% of the rearrangements should be in-frame. In contrast, functional beta selection with no allelic exclusion predicts that 60% of rearrangements should be in-frame, whereas functional beta selection with allelic exclusion predicts that 72% of rearrangements should be in-frame. Sequence analysis of cloned coding joints revealed similar percentages of in-frame rearrangements in both DP samples (Fig. 5B, Lat+/+ 78% and LxRC 72%). Although sample size was insufficient to distinguish whether or not allelic exclusion was enforced, the results provide strong support for beta selection in both samples. Thus, DP thymocytes generated by Raf-CAAX signals rely on a functional Tcrb rearrangement despite the interruption of all known signaling pathways downstream of the pre-TCR.


Figure 5
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  		FIGURE 5. beta-selected DP thymocytes in LxRC mice. A, Intracellular TCRbeta staining (upper right panel) was analyzed in DN thymocytes of Rag2–/– mice (dashed line), and in DP thymocytes of Lat+/– (bold line), and LxRC (thin line) mice, gated as shown in panels on the left. B, Sequence analysis of cloned Vbeta12DJbeta2 rearrangements from Lat+/+ (98% purity) and LxRC (90% purity) DP thymocytes. In-frame rearrangements are presented as a fraction of total rearrangements analyzed for each genotype.

 
Allelic inclusion in Lat–/–Raf-CAAX DN thymocytes

The above experiments imply that on a Lat–/– background, the Raf-CAAX transgene synergizes with the pre-TCR to promote DN to DP transition with beta selection. We wondered whether the same synergy could lead to effective feedback inhibition of Tcrb recombination in DN thymocytes and, thus, allelic exclusion. Our previous analysis of LxRC mice (Fig. 4) could not address this possibility because in that model ongoing Tcrb recombination in DN thymocytes is required to assemble a pre-TCR. Therefore, we bred both Raf-CAAX and Tcrb transgenes onto the Lat–/– background and used a PCR strategy to analyze the endogenous Vbeta to DJbeta2 rearrangement (Fig. 6). As shown previously, neither the Raf-CAAX transgene alone nor a Tcrb transgene alone could block Vbeta to DJbeta2 rearrangement on a Lat–/– background. Moreover, the two transgenes were no more effective when tested together. Thus, although Raf-CAAX can synergize with the pre-TCR in Lat–/– mice to enforce feedback inhibition in the DP compartment, it is unable to do so in the DN compartment. These results imply that there are distinct requirements for feedback inhibition in the DN and DP compartments. In addition, the pre-TCR signaling requirements for feedback inhibition in DN thymocytes appear distinct from those involved in beta selection and DP differentiation.


Figure 6
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  		FIGURE 6. Allelic inclusion in LxRC DN thymocytes. PCR analysis of Vbeta13DJbeta2 coding joints in unfractionated thymocytes from Lat+/– and Lat–/– mice in the presence (+) or absence (–) of Raf-CAAX (RC) and Tcrb transgenes. Three-fold serial dilutions (wedges) of genomic DNA were amplified and analyzed by Southern blot. A sample without DNA (–) served as negative control, and Cd14 amplication was used to assess DNA loading.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous studies of constitutively active Raf1 and HRas1 transgenes indicated that they could promote DN to DP differentiation on a recombinase (and hence pre-TCR-)-deficient background, but could not feedback inhibit Tcrb recombination on a recombinase (and pre-TCR-)-sufficient background. Feedback inhibition of Tcrb recombination associated with allelic exclusion must be enforced in both DN and DP thymocytes. However, the previous studies could not address the status of feedback inhibition in DP thymocytes generated solely by the MAPK pathway, since in the genetic models used, recombinase-sufficient DP thymocytes were likely produced in response to the complete panoply of signals downstream of the pre-TCR. Our examination of LxRC mice addressed this shortcoming, because the recombinase-sufficient DP thymocytes generated are absolutely dependent on Raf signaling. Similarly, the DP thymocytes of RxRC mice are absolutely dependent on Raf signaling. Our analysis of these DP thymocyte populations revealed changes in Tcrb locus germline transcription, histone modifications, and recombinase activity that are typical of DP thymocytes generated by a complete pre-TCR signaling cascade. Thus, feedback inhibition at the Tcrb locus is intact in DP thymocytes generated by Raf signaling.

Unexpectedly, we found that beta selection was preserved in LxRC DP thymocytes. We expected a substantial fraction of these DP thymocytes to contain nonfunctional Tcrb rearrangements due to constitutive MAPK activation. Instead we found high levels of intracellular TCRbeta staining, and sequence analysis of VDJbeta coding joints revealed a similar percentage of functional rearrangements in both Lat+/+ and LxRC DP thymocytes. The maintenance of beta selection suggests that thymocytes expressing both pre-TCR and Raf-CAAX at the cell surface have a selective advantage over those expressing Raf-CAAX alone. This occurs despite the fact that Raf-CAAX is capable of inducing DN to DP differentiation when expressed without a pre-TCR in Rag2–/– thymocytes. One possibility is that the pre-TCR may serve a scaffold function to allow for more efficient signaling by Raf-CAAX. Alternatively, Raf-CAAX may synergize with currently unknown LAT-independent pre-TCR signaling pathways to promote more efficient differentiation and survival signals. To date, the only known TCR signaling pathway documented to be activated independently of LAT is the MAPK pathway itself (30). MAPK activation under these circumstances involves the direct recruitment of the adaptor proteins Grb2 and Sos to phosphorylated CD3{zeta} chains, thus bypassing the need for LAT. Therefore, it is possible that additive Raf signals in pre-TCR expressing LxRC mice may lead to the beta selection observed.

We have definitively shown that Raf signals can promote reduced Vbeta accessibility and down-regulation of Vbeta to DJbeta rearrangement within the DP compartment. Thus, at least some developmental changes that are associated with the allelic exclusion program are induced by the MAPK pathway. Interestingly, a recent examination of Ets-1-deficient (Ets-1–/–) thymocytes also suggested a role for the MAPK pathway in Tcrb allelic exclusion (31). Ets-1 is a transcription factor essential for T cell and B cell development. Ets-1 is activated via phosphorylation downstream of calcium signaling or the Ras-Raf-MAPK pathway. Ets-1–/– thymocytes display impaired DN to DP differentiation, decreased proliferation, and an increase in apoptosis. Moreover, they display a loss of allelic exclusion as judged by surface expression of two distinct TCRbeta proteins and the inability of a Tcrb transgene to inhibit Vbeta to DJbeta recombination. The MAPK pathway may therefore contribute to Tcrb allelic exclusion via the activation of Ets-1.

Despite the enforcement of feedback in DP thymocytes, the inability of the Raf-CAAX transgene to extinguish Tcrb recombination suggests that MAPK activation alone is insufficient to fully signal for Tcrb allelic exclusion. This result is consistent with the results of previous studies (23, 32). Our finding that feedback inhibition is maintained in LxRC DP thymocytes argues that the failure of Raf signals to promote allelic exclusion must map to the DN compartment. The delay we observed in the development of normal DP populations in both RxRC and LxRC thymocytes is consistent with inefficient DN to DP differentiation, which could possibly disrupt feedback inhibition in the DN compartment. Alternatively, this phenotype could reflect a defect in proliferation in the context of normal DN to DP differentiation. We note that a constitutively active Rasv12 transgene was previously shown to promote DP differentiation with thymic cellularity identical to that of controls but, like Raf-CAAX, did not extinguish Tcrb recombination in the DN compartment (22). This result suggests that the disruption of allelic exclusion may be independent of either the efficiency of differentiation or the extent of proliferation.

It is now thought that allelic exclusion of Ag receptor loci may be regulated through multiple mechanisms. Reduced accessibility of Vbeta gene segments in DP thymocytes likely contributes to Tcrb feedback inhibition, but evidence suggests it is not the only requirement since feedback inhibition is maintained even for accessible Vbeta segments (9, 10). Additional studies suggest changes in locus conformation or subnuclear positioning as possible mechanisms for allelic exclusion at Ig loci (33, 34, 35, 36, 37, 38, 39). However, these mechanisms are still not sufficient to explain the maintenance of feedback inhibition on an already rearranged Tcrb allele (40). Therefore, enforcement of allelic exclusion likely involves several layers of control in addition to those already described. The fact that any of these events may be enforced in DN thymocytes, in a pre-TCR-dependent but MAPK-independent manner, may explain the failure to block Tcrb recombination in this compartment in the Raf-CAAX model.

Despite this failure, we find that differentiation directed by Raf-CAAX leads to a phenotypically normal DP population displaying the expected inactivation of Vbeta gene segments. Our inability to detect continued Vbeta to DJbeta rearrangements within the DP compartment in LxRC mice suggests that the MAPK pathway contributes to both differentiation and feedback inhibition of Tcrb recombination. We suggest that at least a component of the feedback mechanism is tightly linked to DN to DP differentiation and is itself sufficient to suppress Vbeta to DJbeta recombination in the DP compartment. Additional studies will be required to clarify the distinct components of feedback inhibition that are operative in DN and DP thymocytes.


   Acknowledgments
 
We thank L. Martinek of the Duke University Cancer Center Flow Cytometry Facility for help with flow cytometry and H. Boutrid and L. Esper for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI35748 (to M.S.K.). A.J. was supported by a National Science Foundation Graduate Research Fellowship. Back

2 Address correspondence and reprint requests to Dr. Michael S. Krangel, Department of Immunology, P.O. Box 3010, Duke University Medical Center, Durham NC 27710. E-mail address: krang001{at}mc.duke.edu Back

3 Abbreviations used in this paper: RSS, recombination signal sequence; DN, double negative; Cad, carbamoyltransferase dihydrorotase; DP, double positive; LAT, linker for activation of T cells; PKC, protein kinase C; SLP-76, Src homology 2 domain-containing leukocyte protein. Back

Received for publication February 16, 2006. Accepted for publication March 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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