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. Author manuscript; available in PMC: 2013 Oct 15.
Published in final edited form as: J Immunol. 2012 Sep 14;189(8):4005–4013. doi: 10.4049/jimmunol.1201380

The Importance of the Erk Pathway in the Development of LAT-Mediated Autoimmunity

Deirdre M Fuller, Minghua Zhu, Surapong Koonpaew, Mariana I Nelson, Weiguo Zhang †
PMCID: PMC3548449  NIHMSID: NIHMS403197  PMID: 22984075

Abstract

The ability of the transmembrane adaptor protein LAT to regulate T cell development, activation, survival, and homeostasis depends upon phosphorylation of its multiple tyrosine residues. The mutation of tyrosine 136 on LAT abrogates its interaction with PLC-γ1, causing severe ramifications on TCR-mediated signaling. Mice harboring this mutation, LATY136F mice, have significantly impaired thymocyte development; however, they rapidly develop a fatal lymphoproliferative disease marked by the uncontrolled expansion of Th2-skewed CD4+ T cells, high levels of IgE and IgG1, and autoantibody production. In this study, we assessed the contribution of multiple signaling pathways in LATY136F disease development. The deletion of the critical signaling proteins Gads and RasGRP1 caused a further block in thymocyte development but, over time, could not prevent CD4+ T cell hyperproliferation. Also, restoring signaling through the NF-κB and NFAT pathways was unable to halt the development of disease. However, expression of a constitutively active Raf transgene enhanced lymphoproliferation, indicating a role for the Ras-MAPK pathway in LAT-mediated disease.

Introduction

The indispensable function of LAT (linker for activation of T cells) in TCR (T cell receptor)-mediated signaling has been clearly demonstrated. LAT-deficient Jurkat cells have severely impaired TCR-mediated calcium mobilization, Erk activation, CD69 upregulation, and AP-1- and NFAT-mediated gene transcription (1). Correspondingly, LAT-deficient mice have a complete block at the DN3 stage of thymocyte development and lack peripheral T cells (2). As a transmembrane adaptor protein lacking intrinsic enzymatic activity, the phosphorylation of LAT following receptor ligation is vital for its function. Following TCR engagement, LAT is phosphorylated by Zap70, allowing it to interact with SH2 domain-containing proteins, such as Grb2, Gads, and PLC-γ1 (3). Mutational studies using LAT-deficient Jurkat cells show that the disruption of LAT interaction with Grb2, Gads, and PLC-γ1 inhibits TCR-mediated Ras activation, calcium flux, and NFAT activation (4).

Upon LAT phosphorylation, Gads, which is constitutively bound to the adaptor protein SLP-76, is recruited to the cell membrane (5). Gads−/− mice contain significantly fewer thymocytes; however, T cells are able to develop and populate the periphery, despite exhibiting defective responses to TCR engagement (6). Additionally, the activation of PLC-γ1 by LAT leads to the formation of DAG (diacylglycerol), a molecule critical for the recruitment of RasGRP1 (Ras guanyl releasing protein 1) (6–8). The critical role of RasGRP1 in linking TCR engagement to Ras-MAPK activation is demonstrated in RasGRP1−/− mice, which have impaired thymocyte development and Erk activation (9).

The interaction between PLC-γ1 and LAT depends upon tyrosine 132 in humans and 136 in mice. Mutation of this site in Jurkat cells disrupts calcium mobilization and NFAT activation (4). Analyses of mice harboring a mutation at tyrosine 136, LATY136F mice, reveal the critical role of the LAT-PLC-γ1 interaction (10, 11). LATY136F mice have a severe but incomplete block at the DN3 stage, allowing a small percentage of cells to mature into DP and SP cells. The CD4+ T cells that populate the periphery in these mice are hyperactivated and expand vigorously, causing a fatal autoimmune disease. This disease is Th2-skewed, characterized by tissue eosinophilia and extremely high serum levels of IgE and IgG1. These mice exhibit splenomegaly, lymphadenopathy, and lymphocyte infiltration into the lung, liver, and kidney. While there are virtually no CD8+ T cells in these mice, the CD4+ population is marked by an effector/memory phenotype of CD44hiCD62Llo with severely decreased levels of surface TCRβ. Correspondingly, TCR-mediated LAT and PLC-γ1 phosphorylation and calcium flux are also impaired. A striking characteristic of the LATY136F mutant T cells is the tremendous amount of IL-4 they are able to produce. However, the development of the LATY136F disease is not dependent upon the assumption of a Th2 lineage fate. In the absence of STAT6, a transcription factor necessary for IL-4 production and Th2 differentiation, LATY136F/STAT6−/− mice develop a lymphoproliferative disease of the same timing and severity as LATY136F mice (12).

To further study the autoimmune disease in LATY136F mice, we developed an inducible deletion system using the ERCre recombinase, ERCre+LATf/m mice (13). In order to bypass the severe developmental defects associated with the LATY136F mutation, the floxed wildtype LAT allele allows for proper T cell development but can be deleted in the periphery upon the injection of tamoxifen. After deletion of the floxed wildtype allele, T cells are left with only a copy of the mutated LATY136F allele. Our data show that treatment with tamoxifen for 3–4 weeks incites the development of an autoimmune disease similar to that seen in LATY136F mice. Thus, the Y136F mutation in mature T cells alone can drive the lymphoproliferative disease (13). Interestingly, using the deletion system described above, mice containing LAT completely deleted in mature T cells (ERCre+LATf/− mice) develop a similar autoimmune syndrome, although the disease observed is much less severe than the LATY136F disease (14). Together, the data from analyses of these LAT mutant mice clearly indicate the importance of this adaptor protein in T cell homeostasis.

In this study, we undertook the task of further illuminating the signaling components involved in LAT-mediated disease, using both LATY136F (LATm/m)and ERCre+LATf/m (LATf/m) mice. We tested if the deletion of the LAT signalosome components Gads, which interacts with LAT to recruit the essential cytoplasmic adaptor protein SLP-76, and RasGRP1, a critical Ras GEF (guanine exchange factor) in T cells, would lessen disease severity. We subsequently examined whether restoring the signaling pathways that are disrupted upon the abrogation of the LAT-PLC-γ1 interaction, namely NFAT, NF-κB, and Erk activation, could ameliorate disease development. Our data indicated that the Ras-MAPK pathway plays an important role in the development of LAT-mediated autoimmunity.

Materials and Methods

Mice

For all experiments, age-matched LATm/+ or ERCre+LATf/+ mice were used as controls and are referred to as wildtype (WT). ERCre+LATf/+ and ERCre+LATf/m mice were generated as described previously (13, 15). To generate transgenic mice expressing constitutively active calcineurin, cDNA encoding a mutant form of the calcineurin catalytic subunit (16), in which the calmodulin binding domain and autoinhibitory domain were deleted, was cloned into the p29Δ2(Sal-) vector. Using CnA and human CD2-specific primers, transgenic offspring were screened for the presence of the CnA transgene. Transgenic mice were then mated for subsequent generations with LATm/m mice to generate LATm/+CnA+ and LATm/mCnA+ mice. IKK2ca mice were purchased from The Jackson Laboratory (Bar Harbor, ME). RafTg mice were provided by Dr. Roger Perlmutter (University of Washington). Gads−/− mice were from Dr. Alec Cheng (Washington University) and RasGRP1−/− mice were obtained from Dr. James Stone (University of Alberta). Mice were housed in specific pathogen-free conditions and were used in accordance with National Institutes of Health guidelines. The experiments described here were reviewed and approved by the Duke University Institutional Animal Care Committee.

In vivo deletion of LAT

Tamoxifen (Sigma) was dissolved in corn oil to the final concentration of 10mg/mL. Eight- to ten-week-old mice were injected intraperitoneally with 200μl of tamoxifen on two consecutive days, followed by injection once a week for four to eight weeks. In all instances, LAT deletion, as determined by GFP expression, was greater than 98% in CD4 T cells.

Flow cytometry analysis and antibodies

Fluorescent antibodies used in flow cytometry, such as anti-CD4, -CD8, -CD62L, -CD44, -Foxp3, and –TCRβ, were all purchased from eBioscience or BioLegend. For surface marker staining, single cell suspensions were prepared from thymuses, spleens, or lymph nodes and were blocked with anti-FcγII/III receptor before staining with different antibody mixtures. For intracellular transcription factor staining for Foxp3, cells were prepared as above. Following surface marker staining, cells were fixed and permeabilized according to the manufacturer’s instructions (BD Biosciences). Cells were then stained on ice in the dark with anti-Foxp3 antibody. All FACS data were acquired on FACSCanto (BD Biosciences) and analyzed with FlowJo Software.

Intracellular cytokine production and staining

For intracellular staining of cytokines, cells were isolated and stimulated with PMA (20ng/mL) and ionomycin (0.5μu;g/mL) in RPMI complete for one hour before the addition of Monensin. Cells were left for an additional three hours before being stained for cell surface markers and then fixed and permeabilized per the manufacturer’s instructions (eBioscience). Samples were then stained with intracellular antibodies, such as anti-IFN-γ, -IL-2, and –IL-4 antibodies (eBioscience). Flow cytometry data were acquired on FACSCanto (BD Biosciences) and analyzed with FlowJo software.

Results

LAT-mediated disease development in the absence of Gads

LATm/m thymocytes undergo a partial block in development, with a small number of T cells escaping into the periphery to instigate disease (10, 11). Because the Gads-SLP-76 complex is still able to bind to LAT upon the mutation of tyrosine 136 (4), activation of PLC-γ1 by the proline-rich P1 domain of SLP-76 may still occur in LATm/m T cells (17, 18). Therefore, we hypothesized that LAT-mediated phosphorylation of PLC-γ1 through Gads-SLP-76 could be driving the residual thymocyte development and T cell proliferation. We thus investigated the role of the Gads-SLP-76 complex in the development of LATY136F autoimmunity.

To assess the contribution of Gads to disease development, we crossed Gads−/− with LATm/m to generate Gads−/−LATm/m mice. As shown in Figure 1A, total numbers of thymocytes were reduced in LATm/m, Gads−/−, and Gads−/−LATm/m mice. LATm/m thymocytes had a partial block in development, with a small percentage of cells escaping the DN compartment and maturing to the DP stage (Figure 1B). The deletion of Gads also led to a marked defect in thymocyte development, with an increased percentage of DN cells and reduced DP and SP compartments compared to wildtype mice, as published previously (6). In contrast, LATm/mGads−/− thymocytes had a near-complete block at the DN stage of thymocyte development and contained virtually no DP or SP cells. By 1 month of age, LATm/m mice had splenomegaly and the number of CD4+ T cells in these mice was greatly increased (Figure 1A). Despite a partial block in thymocyte development, LATm/m CD4+ T cells had undergone expansion to account for 23% of splenocytes (Figure 1C). Gads−/− mice were T cell lymphopenic as spleens were 4% CD8+ and ~2% CD4+ T cells. However, due to the severe thymocyte block, LATm/mGads−/− spleens contained less than 1% of both CD8+ and CD4+ cells. These data suggested that in the absence of the LAT-PLC-γ1 interaction, Gads is critical for the emanation of signals in developing T cells.

Figure 1. Severely impaired thymocyte development in LATm/mGads−/− mice.

Figure 1

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(A) Total numbers of thymocytes, splenocytes, and CD4+ T cells in WT, LATm/m, Gads−/−, and LATm/mGads−/− mice. (B) Representative FACS plots of CD4 vs CD8 expression on thymocytes from 4-week-old mice. (C) Surface expression of CD4 and CD8 on splenocytes from 4-week-old mice. Data are representative of three independent experiments using 2–4 mice per genotype.

To bypass the severe thymocyte developmental block observed in LATm/mGads−/− mice, we also used ERCre+LATf/m Gads−/− mice to further analyze the role of Gads in LAT-mediated autoimmunity. ERCre+LATf/+, ERCre+LATf/m, and ERCre+LATf/+Gads−/− were used as controls (Since mice with the floxed LAT allele used in this study also carry the ERCre transgene, we omitted ERCre from the description of mouse genotypes below for simplicity.) These mice were all treated with tamoxifen for 8 weeks to delete the wildtype LAT allele. The deletion of LAT led to splenomegaly and hyperproliferation of CD4+ T cells in LATf/m mice (Figure 2A), as described previously (13). LATf/+Gads−/− spleens were slightly smaller than LATf/+ spleens but LATf/mGads−/− mice displayed severe splenomegaly, with only slightly reduced spleen weights. LATf/m spleens contained large percentages of CD4+ cells (Figure 2B). LATf/+Gads−/− spleens, on the other hand, had reduced percentages of CD4+ and CD8+ T cells. LATf/mGads−/− spleens contained ~25% CD4+ T cells and, like LATf/m spleens, had virtually no CD8+ T cells. Also, the vast majority of CD4+ T cells in LATf/m, LATf/+Gads−/−, and LATf/mGads−/− spleens bore an effector/memory phenotype of CD44hiCD62Llo. The CD4+ T cells in LATf/+Gads−/− mice expressed lower levels of surface TCR than wildtype cells and LATf/mGads−/− CD4+ TCR levels were even further decreased compared to LATf/m T cells (Figure 2C). These data indicated that Gads is not required for the development of LATY136F-mediated autoimmunity.

Figure 2. Development of autoimmune disease in LATf/mGads−/− mice.

Figure 2

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(A–E) Mice were treated with tamoxifen for 8 weeks to induce deletion of the floxed LAT allele before FACS analysis. (A) The numbers of splenocytes and CD4+ T cells from mice of each genotype. (B) Representative FACS plots of CD4 vs CD8 on splenocytes (top), as well as CD44 and CD62L expression on CD4+ splenocytes (bottom). (C) TCRβ surface expression on CD4+ splenocytes from the indicated mice. (D) Splenocytes were harvested and stimulated with P+I with Monensin for 4 hours. Representative FACS plots of splenocytes stained with 7AAD, CD4, CD8, IL-4, and IFN-γ. Data are representative of two independent experiments using 2–5 mice per genotype.

One of the hallmark characteristics of the LATY136F disease is the heightened production of Th2 cytokines, namely IL-4 (10, 11). Thus, we wanted to assess the cytokine production by LATf/m, LATf/+Gads−/−, and LATf/mGads−/− T cells (Fig. 2D). A small percentage of wildtype cells produced IFN-γ upon stimulation. In contrast to LATm/m cells, which produce solely IL-4, about 30% of LATf/m T cells produced IFN-γ and about 10% of LATf/m cells produced both cytokines. Furthermore, an increased percentage of LATf/+Gads−/− CD4+ T cells stained positive for IFN-γ and IL-4 compared to wildtype cells. Noticeably, LATf/mGads−/− T cells completely lost the ability to produce IFN-γ with about 80% of LATf/mGads−/− CD4+ T cells producing IL-4, reminiscent of LATm/m T cells. Thus, while LATf/mGads−/− T cells were similar phenotypically to LATf/m T cells, they harbored distinct differences in their ability to produce cytokines. Together, these results suggested that Gads deficiency does not prevent the development of LAT-mediated autoimmunity.

Ablation of RasGRP1 does not inhibit disease development

Because the deletion of Gads in LATf/m T cells was unable to prevent the development of disease, we next evaluated if disruption of TCR-mediated Ras/Erk signaling through the deletion of RasGRP1 would be able to inhibit CD4+ T cell proliferation in LATm/m mice. RasGRP1, a Ras GEF, serves to activate Ras following PLC-γ1 activation and DAG production(7). The deletion of RasGRP1 in T cells leads to impaired thymocyte development and Erk activation (9). Because RasGRP1 plays such a critical role in mediating Ras/Erk activation in T cells, we hypothesized that its deletion in LATm/m T cells could stymie disease development. To this end, we crossed LATm/m with RasGRP1−/− to generate LATm/mRasGRP1−/− mice.

As seen previously, LATm/m thymocytes were partially blocked during the DN stage of development (Figure 3A). RasGRP1−/− mice had increased DN and DP compartments with very few cells progressing to either the CD4 or CD8 SP stage. Analysis of LATm/mRasGRP1−/− mice revealed that thymocytes had a more severe block in the DN stage than LATm/m mice, with only 7.4% of thymocytes becoming DP cells compared to 26.6% of LATm/m thymocytes. By 1 month of age, LATm/m spleens already contained a significant percentage of CD4+ T cells (Figure 3B). In contrast, RasGRP1−/− spleens had severely reduced CD8+ and CD4+ populations. And, as a result of the impaired thymic development, LATm/mRasGRP1−/− mice contained negligible amounts of splenic T cells.

Figure 3. Thymocyte development is impaired in LATm/mRasGRP1−/− mice.

Figure 3

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(A) The numbers of thymocytes, total splenocytes, and CD4+ T cells in WT, LATm/m, GRP1−/−, and LATm/mGRP1−/− mice. (B–C) FACS plots of CD4 and CD8 expression on thymocytes (B) and splenocytes (C) from 4-week-old mice. Data are representative of four independent experiments analyzing at least 2 mice per genotype.

To bypass the development block and more definitively analyze the role of RasGRP1 in LAT-mediated disease, we also employed LATf/m mice. LATf/+, LATf/m, LATf/+RasGRP1−/−, and LATf/mRasGRP1−/− mice were treated with tamoxifen for eight weeks. Expression of the mutant LAT allele resulted in splenomegaly and increased spleen cell numbers (Figure 4A). LATf/+RasGRP1−/− spleens were comparable if slightly larger than LATf/+ spleens. Notably, LATf/mRasGRP1−/− spleens were similar in size and splenocyte number to those of the mutant mice. LATf/m and LATf/mRasGRP1−/− mice contained similarly high percentages of CD4+ T cells, all of which bore an effector/memory phenotype (Figure 4B). In addition, most of the CD4+ T cells from LATf/mRasGRP1−/− mice produced IL-4 (Figure 4C). These data suggested a minimal role of RasGRP1 in LAT-mediated disease.

Figure 4. Deletion of RasGRP1 does not inhibit disease development.

Figure 4

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Mice were treated with tamoxifen to delete the wildtype LAT allele for eight weeks before analysis (A–B). (A) Spleen weights and the numbers of splenocytes and CD4+ T cells from mice of the genotypes indicated. (B) CD4 vs CD8 FACS profiles of splenocytes. (C) Cytokine production. Splenocytes were stimulated with P+I in the presence of Monensin for 4 hours before intracellular staining. Data are representative of three independent experiments using 2–5 mice per genotype.

We further analyzed aged LATm/mRasGRP1−/− mice to determine if the few T cells found in young mice could instigate disease over time. By four months of age, LATm/mRasGRP1−/− mice developed a disease similar to that in LATm/m mice, although severity varied in individual mice (Figure 5A). While some mice had moderately increased spleen weights and cellularity compared to wildtype mice, other littermates had striking splenomegaly, similar to that of age-matched LATm/m mice. By this age, LATm/m spleens were ~50–80% CD4+ T cells and contained virtually no CD8+ T cells (Figure 5B). RasGRP1−/− spleens contained a similar percentage of CD4+ T cells compared to wildtype spleens but a much-reduced CD8+ compartment. LATm/mRasGRP1−/− mice all had increased, but variable, percentages of CD4+ cells, from ~23–70%. Again, none of these mice contained significant numbers of CD8+ cells. Additionally, almost all CD4+ T cells in LATm/m, RasGRP1−/−, and LATm/mRasGRP1−/− mice were CD62Llo and CD44hi. Similar to LATm/m cells, LATm/mRasGRP1−/− CD4+ T cells produced tremendous amounts of IL-4 (data not shown). From these data, we were able to conclude that the deletion of RasGRP1 significantly impaired thymocyte development but over time was unable to inhibit the uncontrolled proliferation of mutant T cells.

Figure 5. RasGRP1 in LAT-mediated autoimmune disease.

Figure 5

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(A) Spleen weights and the numbers of total splenocytes and CD4+ cells from 4-month-old mice of the indicated genotypes. (B) Representative FACS plots of CD4 and CD8 expression on splenocytes (top) as well as CD44 vs CD62L expression on CD4+ splenocytes (bottom). (C–D). Foxp3 expression in CD4+ T cells by intracellular staining. Data are representative of three independent experiments using 2–3mice per genotype.

We also assessed whether the deletion of RasGRP1 and Gads could affect Treg cell populations. As we previously reported, (19), Foxp3+ cells were nearly absent in LATm/m mice (Figure 5C). RasGRP1−/− mice had similar and even slightly increased percentages of Foxp3+ cells compared to wildtype controls. Foxp3+ T cells were clearly unable to develop in LATm/mRasGRP1−/− mice. Because 4-month-old LATm/mRasGRP1−/− mice developed the disease, we analyzed LATm/mGads−/− mice of a similar age. LATm/mGads−/− mice also developed a similar disease as LATm/m mice (data not shown). Despite a severe block in thymocyte development (Figure 1), there was a significant population of CD4+ T cells (11.7%) in 4-month-old LATm/mGads−/− mice, likely due to tremendous expansion of these cells (Figure 5D). We also assessed if Gads deficiency had any impact on the development of Treg cells. Despite having decreased CD4+ T cell percentages, Gads−/− spleens actually contained a similar percentage of Foxp3+ T cells in the CD4+ compartment compared to wildtype controls. However, LATm/mGads−/− mice, similar to LATm/m mice, almost completely lacked Foxp3+ T cells. These data indicated that, while RasGRP1 and Gads are critical for conventional T cell development, they are not required for the development of Treg cells.

Expression of constitutively active calcineurin does not rescue LAT-mediated disease

Since disease development was not impeded by the deletion of Gads or RasGRP1, we next determined whether reconstituting the signaling pathways downstream of the LAT-PLC-γ1 interaction could help restore proper T cell function. We first assessed the role of calcineurin signaling in LAT-mediated disease as perturbation of the LAT-PLC-γ1 interaction causes LATm/m T cells to have a severe defect in calcium mobilization and NFAT activation (10). Calcineurin is a calcium/calmodulin-dependent phosphatase whose activity is required for the translocation of NFAT to the nucleus and, ultimately, T cell cytokine production and proliferation (20). Thus, we wanted to assess if the expression of constitutively active calcineurin could restore proper T cell development and activation in the LAT-mediated disease.

In order to test if the enforced expression of calcineurin could ameliorate disease development, we generated transgenic mice expressing constitutively active calcineurin under the human CD2 promoter. These mice (CnA+) contained a form of calcineurin in which the calmodulin binding domain and autoinhibitory domain were deleted. These mice were then crossed with LATm/m mice to produce LATm/mCnA+ mice. As previously reported, expression of constitutively active calcineurin impairs thymocyte development (16). Therefore, LATm/mCnA+ mice did not develop disease by three months of age due to a severe block in thymocyte development that prevented T cells from populating the periphery in these mice (data not shown). We crossed mice expressing constitutively active calcineurin onto the LATf/m background to eliminate this developmental defect.

LATf/mCnA+ mice developed disease after four weeks of tamoxifen treatment comparably to LATf/m mice. Like LATf/m mice, they had splenomegaly and lymphadenopathy (data not shown). Analysis of lymph nodes showed that wildtype mice had normal percentages of CD4+ and CD8+ cells, while LATf/m mice contained only CD4+ T cells (Figure 6A). CnA+ mice had reduced percentages of peripheral T cells, although the CD4:CD8 ratio was relatively normal. LATf/mCnA+ mice contained predominantly CD4+ T cells, indicating that restoring NFAT signaling did not prevent the hyperproliferation of mutant T cells. In addition, CD4+CD25+ Treg cells were absent in these mice (data not shown). Together, these data provided sufficient evidence that restoring NFAT signaling did not prevent disease development.

Figure 6. Restoration of NFAT and NF-κB signaling cannot suppress the LAT-mediated disease.

Figure 6

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Mice were treated with tamoxifen for 4 weeks before analysis. (A) Representative FACS plots of CD4 vs CD8 expression on LN cells. (B) Surface expression of CD4 and CD8 on LN cells. The data shown are representative of two independent experiments using 1–2 mice per genotype.

NF-κB activation does not rescue LAT-mediated autoimmunity

While enforced activation of calcineurin did not lessen disease severity, we also examined the role of another critical pathway downstream of the LAT-PLC-γ1 interaction, canonical NF-κB signaling. The NF-κB family members are normally sequestered in the cytoplasm through association with the inhibitory IκB family. Signaling through the TCR, among other stimuli, induces the IKK (IκB kinase) complex to phosphorylate IκB, which is then ubiquitinated and degraded, allowing for nuclear translocation of NF-κB (21). The IKK complex consists of two kinases, IKK1/α and IKK2/β, as well as a regulatory subunit, NF-κB essential modulator (NEMO)/IKKγ. We generated LATf/mIKK2ca (constitutively active IKKβ) mice by crossing LATf/m mice to a strain generated by Sasaki et al. in which a constitutively active form of IKK2 preceded by a loxP-flanked STOP cassette is inserted into the ROSA26 locus. In the presence of Cre, the floxed stop codon is excised, allowing for the expression of constitutively active-IKK2, as well as the bicistronic expression of EGFP (22).

Mice were injected with tamoxifen for four weeks to induce both the deletion of the floxed wildtype LAT allele and the expression of active IKK2. Analysis of thymuses showed that the expression of constitutively active IKK2 did not visibly affect thymocyte development and did not rescue thymocyte development in LATf/m mice (data not shown). Analysis of disease development clearly showed that expression of constitutively active IKK2 did nothing to ameliorate splenomegaly, as LATf/mIKK2ca spleens were similar in size to LATf/m spleens (data not shown). In the periphery, IKK2ca mice contained similar percentages of CD4+ and CD8+ T cells compared to WT mice. LATf/m and LATf/mIKK2ca lymph nodes consisted of very few CD8+ cells and large percentages of CD4+ T cells, most of which expressed an effector-memory phenotype. Treg cells were also absent in these mice (Figure 6B and data not shown). In sum, our data demonstrated that T cell hyperproliferation cannot be corrected by restoration of NFAT or NF-κB signaling.

Constitutive activation of Raf augments LATY136F disease

As the activation of the Ras/Raf/MEK/Erk signaling cascade is a crucial component of TCR-mediated signaling downstream of PLC-γ1 activation, we decided to investigate the role of this pathway in LAT-mediated autoimmunity. We used transgenic mice expressing a constitutively active form of Raf, which harbors a CAAX farnesylation sequence at its C-terminus. The farnesylation sequence directs Raf to cell membranes, imitating Ras-mediated Raf activation, thus causing constitutive activation of Raf/MEK/Erk (23).

We crossed constitutively active Raf (RafTg) mice with LATm/m mice to generate LATm/mRafTg mice. The size of thymi from LATm/mRafTg mice was similar to that of LATm/m mice but the numbers of thymocytes in these mice were drastically reduced (Fig. 7A). Expression of Raf-CAAX in WT mice had no significant effect on thymocyte development (data not shown). While there are very few DP and SP thymocytes in LATm/m mice, we observed virtually no DP cells and ~70% CD4+ cells in LATm/mRafTg thymuses (Figure 7A). It was shown previously that Raf-CAAX was able to drive RAG2−/− thymocytes to develop from DN to DP (24). Thus, it may accelerate LATm/m thymocyte differentiation from DP to SP. It is also possible that increased percentage of CD4+ T cells is a consequence of extensive expansion of LATm/mRafTg T cells. Furthermore, spleens and lymph nodes from LATm/mRafTg mice were tremendously enlarged and splenocyte numbers were greatly increased compared with LATm/m mice (Figure 7B). LATm/mRafTg spleens consisted of incredibly high percentages of CD4+ T cells, similar to and even exceeding the percentage of CD4+ T cells seen in LATm/m spleens (Figure 7C). Expression of Raf-CAAX failed to correct the developmental defect of Treg cells in LATm/m mice as no Foxp3+ T cells were detected by intracellular staining in LATm/m and LATm/mRafTg mice (data not shown). Interestingly, there was a drastic reduction in the number of B cells in spleens and lymph nodes from LATm/mRafTg mice, especially from aged mice. While B cell development in LATm/m mice was largely normal, it was partially blocked in LATm/mRafTg mice (data not shown). Another mark of the LATY136F disease is severe eosinophilia in peripheral lymphoid organs (10), as seen in Figure 7D. LATm/mRafTg lymph nodes had a higher percentage of eosinophils as compared to control and even LATm/m LNs, indicating an aggressive disease. These data demonstrated that the transgenic expression of Raf in the context of the LATY136F mutation actually exacerbated disease development, implying an important role for the Ras/Erk pathway in guiding disease development mediated by the LAT mutation.

Figure 7. Constitutively active Raf augments disease severity.

Figure 7

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(A) The number of thymocytes and FACS plots on thymocytes. (B) Spleens and LNs from mice of the indicated genotype (left) and total splenocyte numbers (right). (C) CD4 vs CD8 expression on splenocytes. (D) Forward scatter vs side scatter analysis of eosinophil populations. The data shown are representative of three independent experiments using 1–2 mice per genotype.

Discussion

From the data gathered here, it was obvious that the absence of Gads or RasGRP1 had a significant effect on LATm/m thymocyte development. Normally, LATm/m thymocytes exhibit an incomplete block in the DN3 stage (10, 11). In these mice, the DP population constitutes ~20–30% of thymocytes and SP compartments are reduced yet distinct. LATm/mGads−/− mice displayed a near-complete block in the DN stage of thymocyte development, with virtually no DP or SP compartments. LATm/mRasGRP1−/− mice also had a more severe block than LATm/m mice with only ~7% of thymocytes progressing to the DP stage. Because LAT retains partial function upon mutation of tyrosine 136, a number of signaling pathways remain intact following the loss of the LAT-PLC-γ1 interaction and allow for a minimal amount of thymocyte differentiation. These pathways seem to act predominantly through Gads, as the ablation of this protein severely hindered thymocyte development. As the deletion of RasGRP1 also increased the developmental block of mutant thymocytes, one or more of these signaling pathways might also utilize RasGRP1. Perhaps the deletion of these proteins diminished or abolished the residual calcium flux and/or MAPK activation in LATm/m thymocytes. Interestingly, despite having virtually no peripheral T cells by about 1 month of age, LATm/mRasGRP1−/− mice developed disease comparably to LATm/m mice by 4 months of age, indicating that the few T cells able to populate the periphery were sufficient to instigate autoimmunity.

Despite the dramatic effect on thymocyte development, LAT-mediated disease as driven by mature T cells in the LATf/m model was not significantly affected by the loss of Gads or RasGRP1. A previous report demonstrated that the uncontrolled hyperproliferation and Th2 effector function of LATY136F T cells is not dependent on MHC class II (25). However, another study examining the disease mediated by mature mutant T cells demonstrated that LATΔ/Y136F cells, which are similar to our ERCre+LATf/m T cells, require MHC class II to initiate disease upon wildtype LAT deletion (26). These data suggest that LAT-mediated disease onset may require TCR engagement whereas the perpetuation of the disease may be TCR-independent. Thus, it seemed probable that continuous tonic signaling through the TCR could be driving the hyperproliferation of mutant T cells. Through the disruption of Gads and RasGRP1, we were able to effectively disrupt TCR-proximal signaling and observe the effect on mutant T cells. Both LATf/mGads−/− and LATf/mRasGRP1−/− CD4+ T cells had decreased surface expression of TCRβ compared to LATf/m cells. Yet, LATf/mGads−/− and LATf/mRasGRP1−/− mice developed disease similarly to LATf/m mice following eight weeks of tamoxifen treatment, suggesting that tonic signaling through the TCR is not driving the lymphoproliferative disease.

In addition to the impaired central tolerance seen in LATm/m mice, results from our lab show that these mice fail to develop peripheral tolerance and indeed lack natural CD4+CD25+ T regulatory cells (19). Adoptive transfer of wildtype Treg cells into neonatal LATm/m mice prevents the development of the autoimmune disease, highlighting the importance of natural Tregs in the suppression of the LATY136F autoimmune syndrome. Studies have demonstrated that c-Rel, a member of the NF-κB family, is needed for thymic Treg differentiation and regulates Foxp3 transcription by directly binding to cis-regulatory elements at the Foxp3 locus (27, 28). Additionally, NFAT may bind to the foxp3 promoter and play a role in TCR-induced Foxp3 expression (29). However, restoration of these signaling pathways in LATf/m mice did not rescue Treg cell development and failed to prevent disease progression, suggesting that defects in these pathways are not the main reason causing the disease in these mice.

From the data detailed in this report, it is apparent that the LAT-mediated autoimmune disease was able to occur independently of RasGRP1 and Gads, two important molecules in TCR proximal signaling. A previous study reported that decreasing Erk activation through the deletion of Bam32, an adaptor protein known to activate MAPK in B cells, lessens the severity of LATY136F disease (30). In contrast to this, the deletion of RasGRP1, despite increasing the thymocyte developmental block, did not impede progression of disease over time. Perhaps, in the context of this disease, the Grb2-Sos complex, which is able to bind directly to the phosphorylated TCR-ζ chain to activate Ras (31), may be able to compensate for the RasGRP1 deficiency and drive T cell expansion. Notably, expression of constitutively active Raf increased disease severity, as indicated by the tremendous spleen size, CD4+ proliferation, and eosinophilia observed in LATm/mRafTg mice. These data support the idea that the Ras/MAPK pathway plays a significant role in guiding LATm/m T cell hyperproliferation. In this context, Raf may serve to enhance positive selection of mutant thymocytes, as Erk activation is known to contribute to positive selection (32, 33). Also, the presence of Raf-CAAX likely led to persistent Erk activation, causing increased proliferation of mutant T cells. Additionally, if T cell proliferation in LATm/m mice is driven by cytokines, it is possible that Raf-CAAX enhanced Ras-MAPK activation downstream of cytokine receptor signaling and, consequently, increased LATm/m T cell proliferation.

In sum, we have shown that deletion of critical TCR signaling components, Gads and RasGRP1, did not ameliorate the LAT-dependent lymphoproliferative syndrome. Moreover, the restoration of signaling pathways immediately downstream of LAT could not rescue the development of the LATY136F disease. In fact, by enhancing MAPK signaling through the expression of constitutively active Raf, we significantly increased the severity of the disease, indicating that the Erk pathway does indeed contribute to the uncontrolled T cell expansion that occurs upon abrogation of the LAT-PLC-γ1 interaction.

Acknowledgments

Funding: The National Institutes of Health grants AI048674 and AI056156. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We thank the Duke University Cancer Center Flow Cytometry, DNA Sequencing, and Transgenic Mouse facilities for their excellent services.

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