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eLife. 2013; 2: e01020.
Published online Dec 12, 2013. doi:  10.7554/eLife.01020
PMCID: PMC3858598

Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies

Shimon Sakaguchi, Reviewing editor
Shimon Sakaguchi, Osaka University, Japan;

Abstract

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1Anaef, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1Anaef mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios+ PD-1+ CD4+ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1Anaef is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1Anaef naïve CD4+ T cells. CD44 expression, CD4+ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1AnaefMtorchino double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1Anaef T cell dysregulation.

DOI: http://dx.doi.org/10.7554/eLife.01020.001

Research organism: Mouse

eLife digest

Our DNA contains more than three billion nucleotides. Each of these nucleotides can be an A, C, G or T, and groups of three neighboring nucleotides within our DNA are used to represent the 20 amino acids that are used to make proteins. This means that changing just one nucleotide can cause one amino acid to be replaced by a different amino acid in the protein encoded by that stretch of DNA: AAA and AAG code for the amino acid lysine, for example, but AAC and AAT code for asparagine. Known as missense gene variants, these changes can also increase or decrease the expression of the gene.

Every person has thousands of missense gene variants, including about 12,000 inherited from their parents. Sometimes these variants have no consequence, but they can be harmful if replacing the correct amino acid with a different amino acid prevents the protein from performing an important task. In particular, missense gene variants in genes that encode immune system proteins are likely to play a role in diseases of the immune system. For example, variants near a gene called Rasgrp1 have been linked to two autoimmune diseases – type 1 diabetes and Graves’ disease—in which the immune system mistakenly attacks the body’s own cells and tissues.

Now Daley et al. have shed new light on the mechanism by which a missense gene variant in Rasgrp1 can cause autoimmune diseases. Mice with this mutation show signs of autoimmune disease, but their T cells—white blood cells that have a central role in the immune system – develop normally despite this mutation. Instead, Daley et al. found that a specific type of T cell, called T helper cells, accumulated in large numbers in the mutant mice and stimulated cells of a third type—immune cells called B cells—to produce autoantibodies. The production of autoantibodies is a common feature of autoimmune diseases.

Daley et al. traced the origins of the T helper cells to excessive activity on a signaling pathway that involves a protein called mTOR, and went on to show that treatment with the drug rapamycin counteracted the accumulation of the T helper cells and reduced the level of autoimmune activity. In addition to exemplifying how changing just one amino acid change can have a profound effect, the work of Daley et al. is an attractive model for exploring how missense gene variants in people can contribute to autoimmune diseases.

DOI: http://dx.doi.org/10.7554/eLife.01020.002

Introduction

Positive and negative selection of thymocytes generates a population of T lymphocytes with a broad spectrum of antigen-specific T cell receptors (TCR) (Starr et al., 2003; Kortum et al., 2013). It was recognized early on that the small GTPase Ras plays a role (Swan et al., 1995). Three Ras guanine exchange factor (RasGEF) families can activate Ras: SOS, RasGRP, and RasGRF (Stone, 2011). Following TCR engagement, Son of Sevenless (SOS)-1 and -2 are recruited to the plasma membrane via a Grb2-phospho-LAT interaction. Simultaneously, the second messenger diacylglycerol (DAG), generated via PLCγ, directly recruits Ras guanine nucleotide releasing protein 1 (Rasgrp1) to the plasma membrane (Ebinu et al., 1998). Biochemically, Rasgrp1 and SOS1 synergize to induce high-level Ras activation (Roose et al., 2007) and Rasgrp1 serves a critical role in priming SOS1 via Rasgrp1-produced RasGTP (Das et al., 2009). Consequentially, thymocyte development is severely impaired in Rasgrp1-deficient mice (Dower et al., 2000), and not compensated for by SOS RasGEFs. Additionally, there is only minimal compensation for loss of Rasgrp1 coming from Rasgrp3 or Rasgrp4 (Zhu et al., 2012; Golec et al., 2013). Rasgrp1-deficient mice exhibit a strong defect in positive selection and impaired ERK phosphorylation in thymocytes (Dower et al., 2000; Priatel et al., 2002). The importance of the canonical Rasgrp1-RasGTP-RAF-MEK-ERK pathway for developing thymocytes is further underscored by impaired positive selection in ERK-1 and -2 doubly deficient mice (Fischer et al., 2005).

Although Rasgrp1 plays a critical role in the activation of Ras, relatively little is known about its regulation in T lymphocytes or the in vivo importance of such regulation. In addition to membrane recruitment via its DAG-binding C1 domain (Ebinu et al., 1998), Rasgrp1’s GEF activity is enhanced by inducible phosphorylation of threonine 184 (Roose et al., 2005; Zheng et al., 2005). Phospholipase C γ (PLCγ) not only generates DAG but also inositol 1,4,5-trisphosphate (IP3), which binds to IP3 receptors on the endoplasmic reticulum to activate the calcium pathway (Feske, 2007). Interestingly, Rasgrp1 also contains a pair of EF hands, motifs that often bind calcium, which induces conformational changes (Gifford et al., 2007). Rasgrp1 has been reported to bind calcium in vitro (Ebinu et al., 1998). In chicken DT40 B cells, the first EF1 domain enables the recruitment function of a C-terminal PT domain (plasma membrane targeting domain) that cooperates with the C1 domain to recruit Rasgrp1 to the membrane (Tazmini et al., 2009). Notably, the PT domain contribution is substantial in BCR-stimulated B cell lines, very modest in T cell lines, and negligible in fibroblasts (Beaulieu et al., 2007). Genetic deletion of Rasgrp1’s 200 C-terminal amino acids reduces the formation of mature thymocytes in Rasgrp1d/d mice (Fuller et al., 2012). Our recent structural studies revealed that Rasgrp1’s C terminus contains a coiled-coil dimerization domain (Iwig et al., 2013). Rasgrp1 dimerization plays an important role in controlling Rasgrp1’s activity; the second EF hand of one Rasgrp1 molecule packs against the C1 domain of a second molecule in a manner that is incompatible with DAG-binding whereas calcium binding to the first EF hand is predicted to unlock this autoinhibitory dimer interface (Iwig et al., 2013). Lastly, it is unknown if Rasgrp1 may signal to pathways other than the canonical Rasgrp1-Ras-RAF-MEK-ERK cascade, although a link between Rasgrp1 and mTOR (mechanistic target of rapamycin) signaling has been proposed (Gorentla et al., 2011).

Older Rasgrp1-deficient (Coughlin et al., 2005) and Rasgrp1d/d mice (Fuller et al., 2012) develop splenomegaly and autoantibodies. In these mouse models, the complete deletion or truncation of Rasgrp1 greatly decreases T cell development in the thymus (Dower et al., 2000; Fuller et al., 2012), resulting in peripheral T cell lymphopenia followed by accumulation of CD44hi CD62Llo CD4+ T cells (Priatel et al., 2007; Fuller et al., 2012). Autoimmune phenotypes caused by these mutations have been attributed to compromised T cell selection in the thymus and compensatory expansion of peripheral T cells in response to lymphopenia and/or chronic infection. Hypomorphic missense alleles of the signaling molecules ZAP-70 and LAT also impair T cell development in the thymus and culminate in severe peripheral immune dysregulation. For example, an SKG allele of the kinase ZAP-70 has reduced binding-affinity for phospho-TCRζ and leads to autoimmune arthritis in mice (Sakaguchi et al., 2003). Point mutations in ZAP70’s catalytic domain that reduce kinase activity to intermediate levels diminish thymic deletion and Foxp3+ Treg differentiation but preserve peripheral T cell activation, resulting in autoantibody formation and hyper-IgE production (Siggs et al., 2007). Mutation of a single tyrosine in LAT (LATY136F) results in hyperproliferative lymphocytes of a TH2 type (Aguado et al., 2002; Sommers et al., 2002). In each of these cases, peripheral T cell dysregulation is tied to, and potentially explained by, profound deficits in thymic T cell formation.

Single nucleotide variants that cause amino acid substitutions (missense variants; SNVs) or modify the level of gene expression rather than knocking out protein expression are a major form of human genetic variation: most people inherit ~12,000 missense gene variants (The 1000 Genomes Project Consortium, 2010). Given the emerging examples of missense alleles having very different immunological consequences from null alleles, mouse models that analyze the consequences of missense variants in key immune genes are needed to understand the pathogenesis of complex human immune diseases. Common tag SNVs near RASGRP1 are associated with susceptibility to autoimmune (Type 1) diabetes and to thyroid autoantibodies in Graves’ disease (Qu et al., 2009; Plagnol et al., 2011), while 13 unstudied RASGRP1 missense SNVs are currently listed in public databases. A fruitful approach for identifying missense gene variants that dysregulate immune function has been through N-ethyl-N-nitrosourea (ENU) mutagenesis (Nelms and Goodnow, 2001). Here we describe the analysis of a novel ENU-induced missense variant, Rasgrp1Anaef that reveals an important in vivo regulatory function of Rasgrp1’s EF hands. Rasgrp1Anaef is distinct from previously described autoimmune mutations in Rasgrp1, Zap70 or Lat, as Rasgrp1Anaef has no detectable effect on thymocyte development in mice with normal TCR repertoires, but results in peripheral accumulation of a distinct population of Helios+ PD-1+ T-helper cells and production of anti-nuclear autoantibodies. In contrast to Rasgrp1 deletion, the Rasgrp1Anaef missense variant increases tonic mTOR signaling in naïve CD4+ T cells. Genetic reduction of mTOR function in Rasgrp1Anaef mice normalizes CD44 expression on naïve CD4+ T cells and abolishes excessive accumulation of effector T cells and autoantibodies, demonstrating a central role for increased mTOR activity in driving immune dysregulation in Rasgrp1Anaef mice.

Results

Identification of the Rasgrp1Anaef mouse strain with a mutated EF hand in Rasgrp1

As part of a mouse genome-wide screen for immune phenotypes induced by ENU mutagenesis (Nelms and Goodnow, 2001), we identified a variant C57BL/6 (B6) pedigree displaying elevated frequencies of CD44hi CD4+ cells (Figure 1A), elevated CD44 expression on naïve FOXP3 CD44lo CD4+ cells (Figure 1B) and antinuclear antibodies (ANAs) staining with a homogeneous nuclear pattern (Figure 1C,D). The elevated frequency of CD44hi cells trait, which occurred at a frequency consistent with inheritance of a recessive gene variant (Figure 1A), was used to map the mutation in an F2 intercross to an interval between 114 and 121.2 Mb on chromosome 2 (Figure 1—figure supplement 1A).

Figure 1.
An ENU mouse mutant with increased CD44hi CD4 cells and anti-nuclear autoantibodies.

Sequencing of the exons of Rasgrp1, the only gene within this interval with a known immune function, identified an A to G missense mutation in codon 519 within exon 13 (Figure 2A). Whole-exome capture, sequencing and computational analysis of DNA from an affected mouse (Andrews et al., 2012) identified this mutation as the only novel single-nucleotide variant within the interval of interest on chromosome 2 (data not shown). The mutant codon, located in Rasgrp1’s second EF hand (EF2), encodes a neutral amino acid glycine (G) instead of the normal arginine (R), a large polar molecule with a positive charge (Rasgrp1 R519G; Figure 2A,B). Rasgrp1’s arginine residue at 519 is also found in Rasgrp-2, and -4, and in EF3 of calcium and integrin binding protein (CIB) (Figure 2B). EF hands typically come in pairs separated by a linker and calcium binding subsequently alters the angle between helices E and F in proteins such as calmodulin (CaM) (Figure 2C) (Grabarek, 2006; Gifford et al., 2007). Unique to Rasgrp1, this linker is unusually short. Furthermore, biophysical studies revealed that Rasgrp1’s EF2 does not bind calcium, that the E helix is non-existent in EF2, but instead has evolved as a critical loop forming an autoinhibitory interface with the C1 domain (Figure 2D,E) (Iwig et al., 2013).

Figure 2.
Mapping of the ENU mutation to the autoinhibitory second EF hand domain in Rasgrp1.

The ENU-generated allele was named ‘Rasgrp1Anaef’ to reflect the combination of antinuclear antibody (ANA) production and the amino acid substitution in the EF hand. Genotyping of this mutation in multiple generations of B6 offspring (Figure 1—figure supplement 1B,C) demonstrated that inheritance of the Rasgrp1Anaef allele was well correlated with the immunological abnormalities described above and below. ANAs were present in 70% of homozygous Rasgrp1Anaef/Anaef mice and 35% of heterozygous Rasgrp1Anaef/+ mice (Figure 1D), compared to 5% of wildtype B6 mice, indicating a gene dosage effect. The R519G substitution caused an approximate 40% decrease in Rasgrp1 protein levels in homozygous Rasgrp1Anaef/Anaef thymocytes (Figure 2F). Since heterozygous Rasgrp1+/− thymocytes express half the Rasgrp1 dosage (Figure 2G) but do not display an abnormal immune phenotype (Dower et al., 2000), whereas heterozygous Rasgrp1Anaef/+ mice do exhibit abnormal immune phenotypes (Figure 1B,D), we conclude that the immune dysregulation in mice bearing the Rasgrp1Anaef allele is caused by the specific R519G alteration and not simply by a reduction of Rasgrp1 protein levels. In the remainder of the manuscript we discuss the analysis of homozygous Rasgrp1Anaef/Anaef mice and refer to these as Rasgrp1Anaef mice.

Rasgrp1Anaef preserves Rasgrp1 function for T cell selection in the thymus

Analysis of Rasgrp1Anaef mice revealed a striking contrast to the published Rasgrp1-deficient and Rasgrp1d/d mouse models, which have T cell developmental defects that result in low thymic T cell output (Dower et al., 2000; Priatel et al., 2007; Fuller et al., 2012). The frequency and number of mature CD4+ and CD8+ single positive (SP) thymocyte subsets was normal in Rasgrp1Anaef mice, whereas these subsets were markedly decreased in Rasgrp1−/− (knockout) mice analyzed in parallel (Figure 3A,B). Unlike the knockout allele, the Rasgrp1Anaef mutation did not decrease the frequency of CD69hi TCRβhi cells amongst CD4+CD8+ double positive (DP) thymocytes (Figure 3C) or the number of Foxp3+ CD4SP cells in the thymus (Figure 3B). Even in bone marrow chimeras reconstituted with a mixture of CD45.2+ Rasgrp1Anaef and CD45.1+ wildtype marrow, the Rasgrp1Anaef thymocytes exhibited no competitive disadvantage as they matured from DP to SP cells (Figure 3D). Injection into mice of 5-bromo-2′-deoxyuridine (BrdU) to pulse-label a cohort of proliferating DP thymocytes followed by analysis on day 5 demonstrated that the kinetics of maturation into SP cells was unaffected by the Rasgrp1Anaef mutation (Figure 3E, Figure 3—figure supplement 1A). There was also normal deletion of Vβ5+ and Vβ11+ SP thymocytes upon self-superantigen/I-Ek recognition in B10.Br mice (Figure 3F) and similar usage of TCRα Jα segments in wildtype and Rasgrp1Anaef CD4SP thymocyte populations (Figure 3G, Figure 3—figure supplement 1B). Thus, analysis of the thymus of Rasgrp1Anaef mice with a diverse TCR repertoire revealed no abnormalities in positive selection, Foxp3+ T-regulatory (T-reg) cell differentiation or clonal deletion, in striking contrast to previously described Rasgrp1 mutations.

Figure 3.
Rasgrp1Anaef differs from Rasgrp1null by preserving T cell selection in the thymus.

Anaef diminishes canonical Rasgrp1-Ras-ERK signaling in response to in vitro stimulation

Despite the normal thymic development in Rasgrp1Anaef animals, there was a striking biochemical effect of the Rasgrp1Anaef mutation on activation of the canonical Rasgrp1-Ras-ERK signaling pathway in a range of in vitro stimulation assays. GFP-tagged wildtype- or Anaef- Rasgrp1 was transiently expressed in RasGRP1-deficient Jurkat cells (JPRM441) (Roose et al., 2005), which were either left unstimulated or stimulated with a combination of PMA (a synthetic analog of diacylglycerol) and ionomycin (a calcium ionophore). Gating on cells with different GFP intensities (Figure 4—figure supplement 1A) revealed that Rasgrp1Anaef was hypomorphic (partial loss of function) for activating the Ras-ERK pathway: in GFP+ cells expressing Rasgrp1Anaef there was only low ERK phosphorylation (P-ERK) and this was only modestly increased when PMA and ionomycin were added (Figure 4A). By contrast, GFP+ cells expressing high levels of wildtype Rasgrp1 vector induced 5-times higher P-ERK spontaneously and this was doubled by PMA and ionomycin stimulation. Next, we stably reconstituted the JPRM441 cell line, which expresses ~10% of residual wildtype RasGRP1 protein (Roose et al., 2005) with Rasgrp1Anaef or Rasgrp1wildtype vectors and selected clones with Rasgrp1 expression levels similar to the parental Jurkat cell line (Figure 4—figure supplement 1B). Since JPRM441 cells do not express surface TCR (Roose et al., 2005), clonal cell lines were stimulated with PMA followed by RasGTP pull-down assays, which demonstrated that Rasgrp1Anaef decreased PMA-induced GTP-loading of Ras to levels below that of the nontransfected JPRM441 cells (Figure 4B). In the same transfected cell lines, PMA-induced P-ERK responses were decreased in Rasgrp1Anaef expressing cells, most notable with the lower dose of PMA (PMA MED; 5 ng/ml) and contrasted the effective induction of P-ERK signals in Jurkat and JPRM441-WT-Rasgrp1 cells (Figure 4C,D). Similarly, Rasgrp1Anaef expressing cells demonstrated less potent synergy in P-ERK levels when ionomycin was combined with a very low PMA stimulus (2 ng/ml) (Figure 4E, Figure 4—figure supplement 1C). In fact, P-ERK responses in JPRM441-Rasgrp1Anaef cells were more impaired than in the parental JPRM441 cells, indicating a dominant negative effect, which was also observed at the level of Ras activation (Figure 4B). We previously reported a dominant negative effect for ΔDAG-Rasgrp1, a form of Rasgrp1 lacking the DAG-binding C1 domain (ΔDAG) and we postulated that there may be competition with the residual ~10% of wildtype RasGRP1 (Roose et al., 2005). As Rasgrp1 is regulated by DAG-driven membrane recruitment (Ebinu et al., 1998; Roose et al., 2005) we examined this process for Rasgrp1Anaef. EGFP-tagged wildtype or Anaef Rasgrp1-transfected JPRM441 cells were FACS sorted on low GFP expression to avoid overexpression artifacts and cells were allowed to adhere to coated slides. PMA stimulation resulted in membrane recruitment and cytoplasmic clearing of wildtype Rasgrp1; whereas these events were decreased for Rasgrp1Anaef (Figure 4F).

Figure 4.
Rasgrp1Anaef diminishes in vitro signaling to Ras and ERK.

To test TCR-induced Ras-ERK signaling in thymocytes, we first stimulated thymocytes from wildtype or Rasgrp1Anaef mice with anti-CD3 crosslinking antibodies and probed lysates for tyrosine-phosphorylated proteins to examine the global biochemical effects of the Rasgrp1Anaef mutation. Both thymocyte populations demonstrated similar induction of total phospho-tyrosine patterns and similar activating phosphorylation of Lck and Zap-70 that lie upstream of Rasgrp1 (Figure 5—figure supplement 1A). Rasgrp1’s GEF activity is also enhanced by phosphorylation on T184 (Roose et al., 2005; Zheng et al., 2005). Using a new monoclonal antibody specific for P-T184-Rasgrp1 (Figure 5—figure supplement 1B) we observed drastically impaired phosphorylation of T184-Rasgrp1, and reduced ERK phosphorylation in Rasgrp1Anaef thymocytes (Figure 5A). By contrast, Rasgrp1+/− thymocytes heterozygous for the null allele displayed readily detectable Rasgrp1- and ERK- phosphorylation (Figure 5B), demonstrating that the signaling defects in the Rasgrp1Anaef thymocytes are much greater than when the amount of Rasgrp1 is simply halved. Thymocyte subset-specific P-ERK analyses revealed reduced anti-CD3- and PMA-induced responses in DP, CD4SP, and CD8SP Rasgrp1Anaef thymocytes (Figure 5C, Figure 5—figure supplement 1C), echoing the cell line conclusion that the Anaef mutation results in a partial loss of function with respect to induced Ras-ERK signaling. When pressure was placed on TCR-Ras-ERK signaling for positive selection in vivo, by introducing any one of three different rearranged TCR transgenes that are prematurely expressed at higher than normal levels on DP thymocytes, a small decrease in positive selection was revealed in Rasgrp1Anaef TCR-transgenic thymocytes compared to their wildtype controls (Figure 5—figure supplement 2). Collectively, these results lead to the surprising conclusion that the low affinity pMHC stimulation that drives physiological positive selection in vivo is remarkably robust to decreased Rasgrp1 activation of Ras-ERK.

Figure 5.
Rasgrp1Anaef signaling characteristics of in vitro stimulated thymocytes.

Intrinsically dysregulated formation of Helios+ PD-1+ CD4+ T cells in Rasgrp1Anaef mice

Given the evidence above for normal thymic formation of T cells in Rasgrp1Anaef animals with normal TCR genes, we sought to define the peripheral CD4+ cell dysregulation that results in an expanded population of CD44hi CD4+ T cells. Total splenocyte numbers and CD4 subsets were within the normal range in young Rasgrp1Anaef animals, but between 50 and 150 days of age the frequencies of activated or memory CD44hi Foxp3 CD4+ cells increased, as did Foxp3+ CD4 cells, while the frequency of CD44low Foxp3 naïve CD4+ cells decreased (Figure 6A–C).

Figure 6.
Rasgrp1Anaef results in dysregulation of peripheral CD4 T cells.

Further resolution of CD4+ subsets based on intracellular cytokine staining revealed that interferon-γ producing cells were increased in frequency by a similar magnitude as CD44hi cells as a whole (Figure 6D and data not shown). Thus, there was no evidence that the Rasgrp1Anaef mutation skewed T-helper cells towards a Th1 phenotype, but simply increased the number of activated or memory/effector CD4+ cells. Staining for PD-1 and CXCR5, whose high expression on CD4+ cells identifies T follicular helper (TFH) cells (Ramiscal and Vinuesa, 2013) revealed a dramatic expansion of these cells in Rasgrp1Anaef mice (Figure 6E). However most of the increase in CD44hi Foxp3 CD4+ cells in Rasgrp1Anaef mice was due to a 600% increase in cells that expressed intermediate levels of PD-1 and CXCR5, and hence are unlikely to be TFH cells, but were distinguished by high expression of the Helios transcription factor (Figure 6E). Helios is highly expressed in Foxp3+ T-reg cells (Thornton et al., 2010), but in wildtype and Rasgrp1Anaef mice Helios is also upregulated in a subset of Foxp3 CD4+ cells, nearly all of which are CD44hi (Figure 6F). Rasgrp1Anaef greatly increased this Helios+ CD44hi Foxp3 CD4+ population, which was also distinguished by high PD-1 expression in Rasgrp1Anaef (Figure 6F). Rasgrp1Anaef mice had normal frequencies of CD95 (Fas)+ GL-7+ germinal center B cells in the spleen (Figure 6G), consistent with the conclusion that the accumulating Helios+ PD-1+ CXCR5int Foxp3 CD44hi CD4+ cells were a distinct type of activated CD4+ cell but not fully differentiated TFH cells.

To test if the dysregulated accumulation of Helios+ PD-1+ CXCR5int CD44hi CD4+ T cells in Anaef mice required B cells, Rasgrp1Anaef animals were intercrossed with mice bearing a null mutation in the BCR subunit, CD79a (Yabas et al., 2011). In Rasgrp1Anaef Cd79anull animals lacking B cells, accumulation of PD-1+ Helios+ CD4+ T cells was profoundly suppressed (Figure 7A,B). Indeed, the Rasgrp1Anaef–driven distortion in relative frequencies of naïve, effector/memory and regulatory subsets of CD4 splenocytes was rectified by the absence of B cells in Rasgrp1Anaef Cd79anull mice (Figure 7C,D). By contrast, the elevated CD44 expression on naïve CD4+ cells was still present (Figure 7E) indicating this is a constitutive effect of the Rasgrp1Anaef mutation.

Figure 7.
Role of B and T cells in Rasgrp1Anaef-induced Helios+ PD-1+ CD4+ T cell and autoantibody formation.

The requirement for B cells could indicate they are needed as specialized antigen presenting cells, as is the case for TFH cells (Ramiscal and Vinuesa, 2013), or that the Anaef mutation also acts in B cells since B cells also express Rasgrp1 (Stone, 2011). To resolve these alternatives, we used bone marrow from Rasgrp1Anaef Cd79anull animals mixed with wildtype Rasgrp1+/+ marrow to generate chimeric mice where the Rasgrp1Anaef mutation was excluded from B cells but present in most of the T cells (experimental group B in Figure 7F–I), and compared these with control chimeras where all hematopoietic cells were Rasgrp1Anaef or Rasgrp1WT (groups A, C and D in Figure 7). Despite having the Anaef mutation in the T but not B cells of group B mice, a high proportion developed antinuclear autoantibodies comparable to the control group D where both B and T cells carried the Anaef mutation (Figure 7G). Moreover, a high frequency and number of CD45.2+ Rasgrp1Anaef CD4+ T cells acquired a Helios+ PD-1+ phenotype in Group B animals, unlike the co-resident CD45.1+ wildtype T cells (Figure 7I). The accumulation of these activated CD4 T cells thus reflects a cell-autonomous effect of the Anaef mutation within the CD4 T cells and does not depend upon the Anaef allele being present in B cells.

CD44 expression is a sensitive reporter of mTOR activity in naïve T cells

Increased basal expression of the cell adhesion receptor, CD44, was a unique trait exhibited by naïve, CD62L-positive Rasgrp1Anaef T cells (Figure 1B). CD44 expression normally increases during differentiation of DP thymocytes into SP T cells, attains higher levels on naïve CD4 T cells than on naïve CD8 cells, and increases further on activated/memory T cells, but little was known about what determines the level of CD44 expressed. In cancer cells CD44 has been described as an mTOR target (Hsieh et al., 2012). In our ongoing peripheral blood screen of ENU mutagenized mouse pedigrees, we identified a strain, chino, with decreased CD44 expression on peripheral CD4+ CD62Lhi cells but relatively normal T cell numbers and subsets (Figure 8A). This unusual phenotype mapped to a single nucleotide change (T to G) in exon 5 of the mechanistic target of rapamycin (Mtor) gene, introducing serine in place of isoleucine at position 205 in the fifth predicted HEAT domain of the protein (Knutson, 2010) (Figure 8B). This mTOR isoleucine residue is entirely conserved from mammals to yeast (Figure 8—figure supplement 1). The mTOR HEAT-repeat domain forms a large superhelical structure that binds RAPTOR to recruit substrates such as S6 kinase for phosphorylation by the mTOR kinase domain (Kim et al., 2002; Adami et al., 2007).

Figure 8.
CD44 is a sensitive reporter of mTOR activity in immature and naïve CD4+ T cells.

As the absence of Mtor is embryonic lethal in mice (Gangloff et al., 2004; Murakami et al., 2004), the fact that Mtorchino/chino (hereafter referred to as Mtorchino) mice are viable but slightly smaller than wildtype (Figure 8C) indicates that the Mtorchino allele retains substantial function. Consistent with a subtle decrease in mTOR activity, TCR-induced phosphorylation of ribosomal protein S6 (P-S6) was modestly decreased but not abolished in Mtorchino CD4+ splenocytes (Figure 8D). The numbers of DP, CD4SP and CD8SP thymocytes were normal in Mtorchino mice (Figure 8E). Whereas expression of CD69, CD5 and TCRβ on DP and SP thymocytes was normal, CD44 expression was decreased on Mtorchino CD4SP and CD8SP thymocytes (Figure 8F). CD44 expression on peripheral blood CD4+ T cells decreased in an Mtorchino allele dose-dependent manner (Figure 8G), demonstrating that CD44 expression is a highly sensitive reporter of small changes in basal mTOR activity in CD4+ T cells. This conclusion is reinforced by supplementary data from two studies: mice with a neo-insertion in an Mtor intron that decreases Mtor mRNA approximately 70% show a similar selective lowering of CD44 on CD4+ T cells (Zhang et al., 2011) and CD44 levels are also reduced on naïve, CD62L-positive T cells that are deficient for the mTOR activator Rheb or deficient for the mTOR-interacting protein Rictor (Delgoffe et al., 2011). Furthermore, we found that pharmacological inhibition of mTOR with rapamycin in mice treated with low doses that avoid toxicity (Coenen et al., 2007; Araki et al., 2009), resulted in a dose-dependent decrease in CD44 expression on wildtype and Rasgrp1Anaef thymocytes (Figure 8H).

Rasgrp1Anaef exaggerates basal mTOR-S6 signaling and CD44 expression in T cells

Our finding that tonic CD44 expression on naïve T cells sensitively reports small changes in mTOR activity prompted further analysis of this pathway in Rasgrp1Anaef T cells. CD44 was elevated on Rasgrp1Anaef DP and SP thymocytes, in diametric contrast to decreased CD44 on these cells in Rasgrp1−/− knockout mice (Figure 9A,B) (Priatel et al., 2007). A putative defect in regulatory T cells cannot explain the increased CD44 expression on naïve Rasgrp1Anaef CD4+ cells, because in mixed chimeras bearing many wild-type Foxp3+ CD4 cells, CD44 expression was still increased on Rasgrp1Anaef but not on co-resident wild-type CD62L+ FOXP3 CD4+ splenocytes (Figure 9C). The cell autonomous increase in CD44 expression was detectable on Rasgrp1Anaef SP and DP thymocytes (Figure 9—figure supplement 1A,B) and even on CD4+ cells expressing a transgenic TCR (Figure 9D). Elevated CD44 expression on Rasgrp1Anaef cells was selective: there were no distinguishable differences between wildtype and Rasgrp1Anaef cells in CD69 and TCRβ, which are markers of cumulative Ras signaling (D’Ambrosio et al., 1994; Genot and Cantrell, 2000; Starr et al., 2003), nor in CD5 expression, a sensitive reporter of TCR affinity and constitutive or tonic TCR signaling (Azzam et al., 1998; Mandl et al., 2013) (Figure 9E, Figure 9—figure supplement 1A,B), and rapamycin treatment in vivo selectively reduced the increased CD44 expression on Rasgrp1Anaef cells (Figure 8H) but did not impact TCR or CD69 expression (data not shown).

Figure 9.
Selective and cell-autonomous increase in CD44 expression in Rasgrp1Anaef CD4+ T cells.

Consistent with the CD44 data, P-S6 levels in unstimulated TCRβlow (pre-selection) DP thymocytes were modestly increased in Rasgrp1Anaef mice whereas they were decreased in cells from Rasgrp1−/− mice (Figure 10A). CD44 levels gradually rise as T cells mature from DP to SP and peripheral naïve T cells, suggesting that CD44 expression may reflect basal or tonic signaling. Such signals can be visualized by constitutive tyrosine-phosphorylation of the TCRζ chains and other proteins (van Oers et al., 1993), basal levels of ZAP70 recruitment to phosphorylated zeta chains (van Oers et al., 1994; Stefanova et al., 2002), and tonic phosphorylation of ERK (Roose et al., 2003; Markegard et al., 2011). Furthermore, these signals dissipate when T cells are rested in vitro in non-stimulatory medium (van Oers et al., 1993; Stefanova et al., 2002). We investigated tonic mTOR–S6 signals and found substantial basal P-S6 levels in freshly isolated lymph node cells that decreased when cells were serum-starved in vitro (Figure 10B). A recent study reported that naive CD4+ T cells display a range of CD5 expression in which the CD5high cells receive most tonic signal input and are most immune reactive (Mandl et al., 2013). We first sorted CD44low naïve CD4+ T cells into the most bright and most dim expression for CD5 and determined that CD5high naïve CD4+ T cells have significantly more P-S6 than their CD5low counterparts (Figure 10C). Next, dividing CD5low and CD5high naïve CD4+ T cells in equal 50–50% splits revealed that basal P-S6 was increased in Rasgrp1Anaef, particularly in the CD5low subset compared to wildtype cells (Figure 10D). The exact origin of tonic signals in T cells and its function being either immune stimulatory or immune suppressive is still an area of debate (Polic et al., 2001; Smith et al., 2001; Bhandoola et al., 2002; Stefanova et al., 2002; Hogquist et al., 2003), but at least part of the tone appears to be generated by low affinity TCR binding to self pMHC (Stefanova et al., 2002). To examine if self-peptide/MHCII recognition plays a role in the increased CD44 expression on Rasgrp1Anaef naïve CD4+ T cells, we adoptively transferred a mixture of wild-type and Rasgrp1Anaef splenocytes into wild-type or MHCII(H2-Aa)–deficient recipient mice (Figure 10—figure supplement 1). Maintenance of CD5 expression on T cells requires contact with self pMHC (Smith et al., 2001; Mandl et al., 2012) and, as expected, CD5 expression on wildtype CD4+ CD62L+ Foxp3 T cells decreased in MHCII-deficient hosts (Figure 10E), consistent with the hypothesis that CD5 is a sensitive reporter of TCR signal strength. By contrast, CD44 levels were similar irrespective of MHCII expression in the adoptive hosts, and CD44 levels remained higher on Rasgrp1Anaef than co-transferred wild-type cells in both contexts (Figure 10E). These data reveal constitutively increased expression of two reporters of mTOR activity in Rasgrp1Anaef naïve CD4+ T cells: the well-established reporter P-S6 and the reporter clarified here, CD44. The fact that the Rasgrp1Anaef–driven increase in CD44 is retained in the absence of MHCII suggests that this tonic signal is at least partially independent of triggering of TCRs by self-pMHC.

Figure 10.
Increased tonic mTOR-S6 signals in Rasgrp1Anaef thymocytes and T cells.

CD44+ Helios+ PD-1+ CD4+ T cell accumulation and autoantibodies in Rasgrp1Anaef mice are corrected by hypomorphic mTOR mutation

To test the role of elevated mTOR signaling in the Rasgrp1Anaef–induced overexpression of CD44 in naïve T cells and in the accumulation of activated CD44hi PD-1+ CD4+ cells and autoantibodies, Rasgrp1Anaef mice were intercrossed with the subtle loss-of-function Mtorchino strain. Using a CD62L+ Foxp3 gate to resolve naïve CD4+ splenocytes, we found that the Mtorchino mutation abolished the Rasgrp1Anaef–driven increase in CD44 expression in these naïve T cells (Figure 11A). The Mtorchino mutation alone resulted in a decrease in numbers of splenocytes, including CD4+ cells, as was observed in mice with reduced Mtor mRNA (Zhang et al., 2011), but the relative proportions of the CD4+ subsets examined were normal (Figure 11B). Whereas Rasgrp1Anaef mice with normal mTOR accumulated a high frequency of CD44hi Foxp3 CD4+ splenocytes, including the prominent PD-1+ Helios+ subset, this was corrected down to normal numbers in Rasgrp1Anaef Mtorchino double mutants (Figure 11B,C). Moreover, in bone marrow chimeras bearing Rasgrp1Anaef Mtorchino double-mutant hematopoietic cells, the frequency of animals with antinuclear autoantibodies was corrected to the low frequency observed in control chimeras with wildtype Rasgrp1 and Mtor (Figure 11D). Collectively, these results establish that accumulation of CD44hi Helios+ PD-1+ CD4+ cells and autoantibodies induced by Rasgrp1Anaef is sensitive to small differences in mTOR signaling.

Figure 11.
Mtor hypomorphic mutation corrects Rasgrp1Anaef–induced increase in naive T-cell CD44 expression and accumulation of CD44+Helios+PD-1+CD4+ cells and autoantibodies.

Discussion

The findings here reveal a new role for Rasgrp1 in the cell-intrinsic regulation of peripheral CD4+ T cells. By analyzing a missense mutation in the Rasgrp1 EF-hand, the results dissociate this new function of Rasgrp1 from its well-known role in thymic positive selection. While the Anaef EF-hand mutation did decrease Rasgrp1 activation of Ras and ERK when thymocytes were stimulated acutely by antibodies to CD3 or with PMA in vitro, the activity of this pathway during physiological positive selection in vivo remained sufficient for normal numbers of single positive thymocytes and peripheral T cells to form even under competitive reconstitution conditions. T cell lymphopenia and sparse T cell repertoires secondary to defective positive selection potentially explain the autoantibodies observed in mice where Rasgrp1 is entirely absent or C-terminally deleted (Dower et al., 2000; Coughlin et al., 2005; Priatel et al., 2007; Fuller et al., 2012), and in animals with missense mutations in ZAP-70 or LAT (Aguado et al., 2002; Sommers et al., 2002; Sakaguchi et al., 2003; Siggs et al., 2007). By contrast, the normal thymic development coupled with experiments in mixed bone marrow chimeras where wild-type and mutant T cells co-exist rules out this possibility for the Rasgrp1Anaef mutation, and shows that peripheral CD4 cells are intrinsically dysregulated. By a combination of biochemical and genetic studies, we identify overactive mTOR signaling within naïve CD4 T cells as a key component for Rasgrp1Anaef to drive two abnormalities: (1) a constitutive increase in CD44 expression in naïve CD4 T cells and (2) a gradual accumulation of peripheral Helios+ CD44hi CD4 cells and autoantibodies.

Rasgrp1Anaef’s effect on Ras/ERK signaling in vivo was much milder than expected from its effects in in vitro assays. In response to relatively strong in vitro stimuli, the Rasgrp1Anaef mutation results in impaired membrane recruitment and T184 phosphorylation of Rasgrp1 as well as reduced activation of Ras-ERK, establishing that Rasgrp1Anaef is a hypomorphic (partial loss-of-function) allele under these conditions. By contrast, in vivo, Rasgrp1Anaef did not alter CD69 and TCRβ induction or positive selection of thymocytes, unlike the C-terminally deleted (Rasgrp1d/d) and knockout (Rasgrp1−/−) alleles which caused moderate and severe decreases in these processes, respectively. This may indicate that the cumulative Ras-ERK signals required for these events are sufficiently buffered or robust that they tolerate a modest reduction in Rasgrp1’s Ras activating activity. Only when a TCR transgene was prematurely expressed at higher than normal levels in DP thymocytes was there a measurable deficit in positive selection, and even under these conditions there was a small decrease in positive selection compared even to the subtle Zap70murdock mutation (Siggs et al., 2007). This is surprising given that the induction of these signals involves low affinity pMHC binding by the TCR, which might be expected to be particularly sensitive to small changes in Ras-ERK signal strength (Kortum et al., 2013).

CD44 expression on thymocytes is decreased in the complete absence of Rasgrp1 (Figure 9) (Priatel et al., 2007) and increased by oncogenic Ras (Kindler et al., 2008; Zhang et al., 2009; Wang et al., 2011) establishing that CD44 expression in thymocytes is positively regulated by Ras. T-cell CD44 expression is also sensitive to mTOR activity, being reduced by the partial loss-of-function Mtorchino (Figure 8) and Mtortm1Lgm (Zhang et al., 2011) alleles, and dramatically decreased in the absence of Rictor (Delgoffe et al., 2011), a binding partner of mTOR. Rasgrp1Anaef thymocytes and naïve CD4 T cells have increased CD44 and P-S6 expression, suggesting that the Anaef mutation increases either Rasgrp1/Ras/ERK signaling or PI-3-kinase/mTOR/S6 signaling, or both. Our recent biophysical studies revealed that Rasgrp1’s EF hands keep the protein in an autoinhibited, dimeric state, and modeling indicates that calcium-binding to the EF domain would relieve autoinhibition (Iwig et al., 2013). Thus, Rasgrp1’s EF hands play both stimulatory- and inhibitory-roles that may result in the EF2 substitution in Rasgrp1Anaef decreasing maintenance of the autoinhibited state in the absence of strong TCR stimuli and decreasing RasGRP1 activation during strong TCR stimulation. Evidence exists for multiple intersections between the RasGRP1/Ras/ERK/RSK and PI-3-kinase/mTOR/S6 pathways, including at the level of Ras with PI-3-kinase (Castellano and Downward, 2010) and at the level of RSK with S6 (Salmond et al., 2009). While the mechanism is currently unclear, the current evidence suggests that Rasgrp1Anaef’s gain-of-function in naïve T cells in the absence of strong TCR stimulation–and apparently in the absence of MHCII ligands for the TCR (Figure 10E)—activates S6-CD44 more than it activates ERK-CD69.

In T cells, the mTOR pathway is activated by strong TCR stimulation (Gorentla et al., 2011) and is required for efficient differentiation of naïve CD4 cells into effector cells (Delgoffe et al., 2009). T-cell-specific deletion of Tsc1, a negative regulator of mTOR, results in increased levels of P-S6 and an exuberant response to TCR stimulation in naïve T cells (Yang et al., 2011). Increased mTOR stimulation by Rasgrp1Anaef may allow self-antigens to activate some naïve CD4 cells, resulting in the gradual accumulation of activated CD62Llow CD44hi PD-1+ HELIOS+ T cells and antinuclear autoantibodies. Because accumulation of PD-1+ HELIOS+ T cells in Rasgrp1Anaef mice requires B cells (Figure 7), these T cells might require B cells as specialized APCs or they might require Fc receptor-dependent enhancement of antigen presentation by antibodies (Silva et al., 2011).

Given the huge number of missense variants in each person (The 1000 Genomes Project Consortium, 2010), patients with autoimmune diseases are more likely to have point mutations in various genes than complete loss of gene expression. This new Rasgrp1Anaef mouse model adds to an emerging category of animal models with point mutations in TCR signaling proteins, along with Zap70skg (Sakaguchi et al., 2003), Zap70murdock and Zap70mrtless hypomorphic alleles (Siggs et al., 2007), LATY136F mice (Aguado et al., 2002; Sommers et al., 2002), and Card11unmodulated mice (Jun et al., 2003), where a partial deficit in T cell signaling precipitates autoimmunity or allergy. RASGRP1 splice variants have been documented for patients with SLE (Yasuda et al., 2007) and abnormal microRNA-driven downregulation of Rasgrp1 expression may play a role in aberrant DNA methylation in Lupus CD4+ T cells (Pan et al., 2010). In addition, RASGRP1-linked SNVs have been associated with autoimmune diabetes and thyroid disease (Qu et al., 2009; Plagnol et al., 2011). Of the 13 uncharacterized RASGRP1 missense SNVs currently known, rs62621817 is of particular interest here since it causes a missense variation in RasGRP1’s first EF hand, changing a conserved, negatively charged aspartic acid into a valine residue. We propose that Rasgrp1Anaef mice may provide a useful model system for further studies to help elucidate how RASGRP1 variants contribute to autoimmune disease, and to help target future efforts to modulate this pathway pharmacologically.

Materials and methods

Mice

Mice were housed in pathogen-free conditions and experiments approved by either the Australian National University Animal Ethics and Experimentation Committee (Goodnow group, A2011/46) or the Institutional Animal Care and Use Committee of the University of California, San Francisco (Roose group, AN084051-01). C57BL/6 (B6), C57BL/6.SJL (CD45.1), B10Br, B10Br.CD45.1, B10Br TCR3A9, Cd79anull (also called Cd79am1ANU) and MHCII-deficient (H2-Aatm1Blt) mice were obtained from ANU Bioscience Services. The Rasgrp1Anaef and Mtorchino strains were established through ethylnitrosourea (ENU)-mediated mutagenesis of B6 mice at the Australian National University as previously described (Randall et al., 2009).

Genetic mapping of the Anaef mutation

Affected Rasgrp1Anaef mice were crossed onto the CBA/J background to generate heterozygous F1 mice. F1 mice were intercrossed to yield mice homozygous for the Anaef mutation and carrying a mix of C57BL/6 and background CBA/J single nucleotide polymorphisms (SNPs). Genomic DNA samples isolated from both affected and unaffected mice were used as templates for SNP mapping at the Genomics Institute of the Novartis Research Foundation (San Diego, CA). SNP markers were spaced approximately every 3–5 Mbp throughout the genome. Once a defined interval was established, the Rasgrp1 encoding gene was sequenced from genomic DNA from both affected Anaef and WT mice. All exons were amplified by PCR with primers designed to include intronic RNA splice donor and acceptor sites. Exome enrichment using the SureSelect Mouse Exome kit (G7550A-001; Agilent, Santa Clara, CA), sequencing using the Illumina HiSeq 2000 (Illumina, San Diego, CA), and computational analysis to detect novel single-nucleotide variants were performed as described previously (Andrews et al., 2012).

Genotyping

Roose lab Anaef mice were genotyped using MS-PCR. Primers were combined in a single reaction with Taq, Taq buffer and dNTPs (all New England BioLabs, Ipswich, MA). Goodnow lab Anaef mice were genotyped by APF Genomics Services following the manufacturer’s instructions for Amplifluor PCR (SNP FAM/JOE; Millipore, Billerica, MA).

Transfections and stable cell lines

Transfections and creation of stable cell lines was performed as previously described (Roose et al., 2005).

Antinuclear antibody testing

Diluted mouse plasma was applied to HEp-2 slides (Inova, San Diego, CA). AlexaFluor488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) was added and slides mounted with fluorescence mounting medium (Dako Australia). Photos were taken using an Olympus IX71 microscope and WIB filter with 20 × lens and exposure time of 1/25 s.

Flow cytometry

Suspensions of splenocytes (depleted of erythrocytes by brief osmotic lysis) or thymocytes were incubated with cocktails of anti-mouse antibodies specific for: CD44, CD4, CD45.1, CD45.2, CD5, CD62L, CD69, TCRβ, PD-1, TCR Vβ5, TCR Vβ8, TCR Vβ11, B220, CD95, GL-7, B220 (BD Pharmingen Franklin Lakes, NJ or BioLegend San Diego, CA) or CD8 (BD Pharmingen and UCSF Monoclonal Antibody Core, clone YTS169.4) conjugated to AlexaFluor700, APC-780 or APCCy7, PE-Cy7, APC, PerCPCy5.5, FITC, PE, Pacific Blue or biotin. Biotinylated antibodies were detected in another incubation step with streptavidin conjugated to Qdot605 (Invitrogen) or BV605 (BioLegend). Cells expressing the 3A9 TCR transgene were detected using the 1G12 (mouse IgG1) antibody (ATCC, Manassas, VA) followed by another incubation in anti-mouseIgG1 (A85.1). To detect intracellular proteins, cells were fixed and permeabilized using a Foxp3 staining kit (eBioscience, San Diego, CA), then labeled with antibodies specific for Foxp3 (FJK-16s; eBioscience), Helios (clone 22F6; Biolegend), IFNγ, IL-4, IL-2 or IL-17 (all BD Pharmingen). Flow cytometry data was acquired on a FACSort (Becton Dickinson) or an LSR Fortessa system and analyzed with FlowJo v8 (Treestar, Ashland, OR).

Cell stimulations

Cells were stimulated using 25 ng/ml (HIGH), 5 ng/ml (MED), or 2 ng/ml (LOW) PMA (Calbiochem) with/without ionomycin (10 μM, Sigma, St. Louis, MO), or with 100 ng/ml PMA for Phospho-S6 assays. To mimic TCR engagement, cells were pre-labeled using anti-CD3 primary antibody (10 μg/ml, UCSF Monoclonal Antibody Core, clone 2C11) and crosslinking was achieved using goat anti-hamster antibody (10 μg/ml, Jackson ImmunoResearch). For intracellular cytokine detection by flow cytometry, splenocytes were stimulated in complete medium for 4 hr at 37°C with PMA (100 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma) and GolgiStop (1/1000; BD), then labeled as described above.

Flow cytometry for phosphorylated proteins

Procedure was performed as described in Das et al., (2009). Cells were fixed using Cytofix Cell Fixative (BD Biosciences). Cells were permeabilized using 90% Methanol. Primary staining for phospho-Erk occurred using rabbit anti-mouse p-Erk antibody (#4377S; Cell Signaling) followed by staining with goat anti-Rabbit PE (Jackson ImmunoResearch, West Grove, PA). For analysis of tonic and PMA induced S6 phosphorylation by FACS, single-cell suspensions were prepared from thymus. Half of the cells were fixed in warm cytofix (BD Biosciences) immediately after harvesting and reserved for tonic signaling analysis. The remaining cells were then counted, and 106 cells/sample were stimulated with PMA for 3 min, followed by fixation. Stimulated and unstimulated cells were then washed three times with cytoperm buffer (BD Biosciences) and incubated on ice in this buffer with a rabbit polyclonal antibody against phosphorylated S6 (cell signaling) for 45 min. The cells were then washed twice with cytoperm and incubated for an additional 45 min in cytoperm buffer containing an APC-conjugated goat anti-rabbit secondary antibody, as well as anti CD8-FITC, CD4, PE-Cy7 and TCRβ-PE. Cells were washed twice and analyzed in an LSR Fortessa system.

Ras pulldown

Activation of Ras was analyzed using a RasGTP pulldown assay (Upstate Biotechnology, Lake Placid, NY) as previously described (Roose et al., 2005).

Western blotting

Cells were lysed using Nonidet-P40 lysis buffer (1%) supplemented with protease and phosphatase inhibitors. Lysates were run on 10% acrylamide Bis-Tris gels and transferred onto PDVF filter (Millipore, Immobilon-P). Blots were probed for Rasgrp1 (M199; Santa Cruz, Dallas, TX), Alpha tubulin (Sigma), phospho-tyrosine (In house antibody prep, clone 4G10), phospho-Zap70 (Y493; Cell Signaling, Danvers, MA), phospho-PLCγ (Y783; Cell Signaling) and phospho-Lck (Y416; Cell Signaling), anti-ERK (pan-Erk) antibody (BD Transduction labs, clone 16/ERK). Rabbit anti-human Rasgrp1 (clone E80) was produced by Epitomics, Inc. (Burlingame, CA, USA). Mouse anti-Rasgrp1 p-T184 (clone JR-pT184RG1-4G7) was produced by AnaSpec (Fremont, CA, USA). Signal from primary antibodies detected using HRP conjugated secondary antibodies: Sheep anti-mouse HRP (GE Healthcare, Cleveland, OH) and goat anti-rabbit HRP (SouthernBiotech, Birmingham, AL). Blots were developed using Pierce ECL Western Blotting Substrate (ThermoScientific, Waltham, MA) and images recorded using a chemiluminescence imager (LAS-4000; Fuji).

Bone marrow chimeras

Bone marrow was collected, and in some experiments, depleted of T cells and NK cells by magnetic labelling using biotinylated anti-TCRβ and anti-NK1.1 and streptavidin microbeads followed by passage through a MACS LD column (Miltenyi Biotec, Germany). Recipient mice were irradiated with X-rays (2 doses of 4.5 Gy given 4 hr apart) then injected intravenously (i.v.) with 2 × 106 bone marrow cells that were either from single or multiple donors as described in the text.

Rapamycin treatment

Suboptimal doses of rapamycin (Coenen et al., 2007; Araki et al., 2009) were prepared on day 0 in sufficient quantity for all injections in one experiment. The appropriate rapamycin stock volume was diluted to the indicated concentrations in DMSO (15.4%), Cremaphor (15.4%) and water (69.2%), and aliquoted in six equal portions (one aliquot per injection) and frozen. Mice were injected on days 0, 1, 2, 3, 5 and 7, and then sacrificed on day 8. Thymocytes were harvested and analyzed as before.

BrdU labelling

1 mg BrdU (BD) in PBS per mouse was injected i.p. to pulse label a cohort of dividing cells. Following surface staining of thymocytes as above, BrdU was detected following the BrdU Flow Kit (BD) protocol by fixing and permeabilizing cells with provided buffers, incubating for 1 hr at 37°C in DNase, then washing and staining with anti-BrdU antibody.

CellTrace Violet (CTV) labelling

CTV labeling was done at room temperature as described (Quah and Parish, 2010) with slight modifications. Splenocytes suspended at 108 cells/ml in RPMI containing HI-FCS (10% vol/vol) were transferred to the base of a fresh 15 ml conical tube. 1 µl of CTV (Life Technologies, Carlsbad, CA) stock solution (10 mM) per ml of cell suspension was placed on the dry wall of the tubes, then tubes were capped, inverted and briefly vortexed (final CTV concentration 10 µM). After 5 min incubation in the dark, 10 ml of 10%FCS/RPMI was added, then cells were sedimented by centrifugation before another wash in 10 ml of the same medium. Cells were then resuspended in PBS and passed through a 70 µm cell strainer (BD) before i.v. injection (200 µl per mouse).

Acknowledgements

The authors would like to thank Drs Richard Glynne, Director of Genetics and Neglected Diseases at Novartis, for guiding the mapping project, Rich Lewis for discussion on calcium signals, and Michelle Hermiston for assistance with thymocyte FACS stainings. We thank Dr Jim Stone for sharing Rasgrp1 deficient mice and Dr Robert Barrington for sending these mice. We thank Debbie Howard, Nadine Barthel and Heather Domaschenz for expert technical assistance, and the animal services and genotyping teams at the Australian Phenomics Facility. We also thank the members of the Cyster, Goodnow, Vinuesa, and Roose labs and NHMRC Program members for helpful comments and suggestions.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

  • Sandler Program in Basic Science to Jeroen P Roose.
  • National Institutes of Health K01CA113367, ARRA supplement, R56-AI095292 to Jeroen P Roose.
  • Wellcome Trust 082030/B/07/Z to Christopher C Goodnow.
  • Department of Innovation, Industry, Science, Research and Tertiary Education to Edward M Bertram, Christopher C Goodnow.
  • Clive and Vera Ramasciotti Foundation Grant to Anselm Enders, Christopher C Goodnow.
  • National Health and Medical Research Council GNT1035858 to Anselm Enders.
  • National Institutes of Health 1R03AR062783-01A1 to Andre Limnander.
  • National Institutes of Health R01-AI74847 to Jason G Cyster.
  • National Institutes of Health R01 AI52127, U54 AI054523 to Edward M Bertram, Christopher C Goodnow.
  • National Health and Medical Research Council Program Grant and Australia Fellowship to Christopher C Goodnow.

Additional information

Competing interests

The authors declare that no competing interests exist.

Author contributions

SRD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

KMC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

DYH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

KLR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

CNJ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

AL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.

DRM, Acquisition of data, Analysis and interpretation of data.

NKP, Acquisition of data, Analysis and interpretation of data.

AE, Acquisition of data, Analysis and interpretation of data.

CR, Acquisition of data, Analysis and interpretation of data.

BB, Acquisition of data, Analysis and interpretation of data.

LAM, Acquisition of data, Analysis and interpretation of data.

GS, Acquisition of data, Analysis and interpretation of data.

EMB, Acquisition of data, Analysis and interpretation of data.

MAF, Acquisition of data, Analysis and interpretation of data.

YS, Acquisition of data, Analysis and interpretation of data.

TDA, Acquisition of data, Analysis and interpretation of data.

BW, Acquisition of data, Analysis and interpretation of data.

SWB, Acquisition of data, Analysis and interpretation of data.

JRW, Acquisition of data, Analysis and interpretation of data.

JGC, Conception and design, Analysis and interpretation of data.

CCG, Conception and design, Analysis and interpretation of data, Drafting or revising the article.

JPR, Conception and design, Analysis and interpretation of data, Drafting or revising the article.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the University of California San Francisco (UCSF; approval number AN084051-03) and of the Australian Phenomics Facility and the Australian National University (ANU; approval number A2011/46). These protocols were approved by the Committee on the Ethics of Animal Experiments of UCSF and ANU.

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eLife. 2013; 2: e01020.
Published online Dec 12, 2013. doi:  10.7554/eLife.01020.022

Decision letter

Shimon Sakaguchi, Reviewing editor
Shimon Sakaguchi, Osaka University, Japan;

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “A missense variant of Rasgrp1 increases tonic mTOR signals resulting in aberrant helper T cells and autoantibodies” for consideration at eLife. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors, and one of whom, Jonathan Powell, has agreed to reveal his identity.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

1) It seems likely that the Rasgrp1Anaef mutation results in the loss of function of the molecules by a dominant negative effect and reduced expression of Rasgrp1 (Figure 2 and Figure 4). However, the mutation enhanced CD44 expression, which is apparently opposite to the phenotype of Rasgrp1null mutation. While the authors claim that a slight difference in mTOR-CD44 pathway would be responsible for the phenotypic difference, more precise analysis is required for addressing the difference. Similarly, tonic mTOR activation and reduced ERK signaling do not link to the observed effects, such as the increase of anti-nuclear antibodies and the accumulation of a CD44hi T cells. In addition, with the finding that CD44, Helios, PD-1, CXCR5, Foxp3 and IFNg were upregulated in Rasgrp1Anaef mice, can these phenotypes also be explained by the slight change of mTOR and the inhibition of ERK? Are there any common mechanisms for the regulation of these genes? Why does a fraction of CD4+ T cells remain CD44lo ? Are those cells expressing a peculiar phenotype when compared to the CD44hi CD4+ T cells?

2) Although the mutant mice showed significant reduction of the ERK signaling, the authors concluded that there were no abnormalities in CD4SP thymocytes, regulatory T cells, and clonal deletion. The authors need to examine whether their functions, such as suppressive activity of regulatory T cells, are normal.

3) In Figure 5A, the reduced levels of P-T184 in the Rasgrp1Anaef thymocytes may be due to the reduced levels of Rasgrp1Anaef protein and to the poor stability of Rasgrp1Anaef following TCR stimulation. To dismiss this possibility, the blot needs to be immune-blotted with an anti-Rasgrp1 antibody (as shown in Figure 2E). In addition, the Rasgrp1Anaef mutation could be more similar to the Rasgrp1null mutation compared with the Rasgrp1+/- mutation. To compare functional differences, it is recommended to include the Rasgrp1null mutation in Figure 5.

4) In Figure 10, the data suggesting that “substantial” levels of P-S6 are found in freshly isolated lymph nodes need to be shown.

5) The conclusion that tonic TCR signal results in basal mTOR-S6 needs to be further substantiated since it is based on the aggregation of disparate data from the Germain laboratory and from the authors’ laboratory.

6) To identify the mutated gene, the authors narrowed down the region where the mutation located and picked up Rasgrp1 because it was the only gene with an immune function within the located region. As some phenotypes were different between Rasgap1null and Rasgap1Anaef mutation, is there a possibility that another mutation locating on the adjacent region is involved in the observed phenotypes? In this context, if the progeny is generated from Rasgrp1Anaef mice that are ANA negative (Figure 1D), are they ANA positive?

7) It might be interesting if the authors quickly review papers that have genetically deleted mTOR signaling in T cells to see if there might be differences in CD44 expression in those mice that might not have been emphasized in the original papers.

eLife. 2013; 2: e01020.
Published online Dec 12, 2013. doi:  10.7554/eLife.01020.023

Author response

1) It seems likely that the Rasgrp1Anaef mutation results in the loss of function of the molecules by a dominant negative effect and reduced expression of Rasgrp1 (Figure 2 and Figure 4). However, the mutation enhanced CD44 expression, which is apparently opposite to the phenotype of Rasgrp1null mutation. While the authors claim that a slight difference in mTOR-CD44 pathway would be responsible for the phenotypic difference, more precise analysis is required for addressing the difference. Similarly, tonic mTOR activation and reduced ERK signaling do not link to the observed effects, such as the increase of anti-nuclear antibodies and the accumulation of a CD44hi T cells. In addition, with the finding that CD44, Helios, PD-1, CXCR5, Foxp3 and IFNg were upregulated in Rasgrp1Anaef mice, can these phenotypes also be explained by the slight change of mTOR and the inhibition of ERK? Are there any common mechanisms for the regulation of these genes? Why does a fraction of CD4+ T cells remain CD44lo ? Are those cells expressing a peculiar phenotype when compared to the CD44hi CD4+ T cells?

We apologize for not being clear about the various consequences of the Anaef mutation in the original manuscript. We have made a number of changes to clarify the evidence that the Anaef mutation has 2 distinct effects: (1) it increases naïve T-cell CD44 expression and (2) it drives accumulation of CD44hi effector CD4+ T cells and autoantibodies.

To distinguish between effects (1) and (2) above, new or modified figure panels demonstrate that Rasgrp1Anaef increases CD44 expression in naïve T cells even in heterozygous Rasgrp1Anaef/+ mice (new Figure 1B), in the absence of B cells (new Figure 7C and 7E) or in the absence of MHCII expression (new Figure 10E), in the presence of large numbers of wildtype Treg cells in mixed chimeras (new Figure 9C) and increased CD44 levels are detectable even at the DP stage of thymocyte development (new Figure 9B and Figure 9–figure supplement 7B). The opposite effects of the null and Anaef alleles on CD44 expression in CD4SP thymocytes suggest that Anaef is in fact a gain-of-function allele under steady-state conditions in vivo. Rasgrp1’s autoinhibitory homodimer conformation recently published in eLife (Iwig et al.) offers an explanation for this: in the absence of calcium, the second EF hand (mutated in Anaef) conceals the DAG-binding C1 domain on the opposing monomer. We now cite papers that demonstrate intersections between the Ras and mTOR signaling pathways and conclude that, although the mechanism is unclear, Rasgrp1Anaef’s gain-of-function activates the mTOR pathway more strongly than it activates the ERK pathway.

The immediacy of effect (1) contrasts with the gradual, age-dependent nature of effect (2), i.e., accumulation of CD44hi CD4+ T cells (Figure 6C). These 2 effects are also separable in that only effect (2) requires B cells (Figure 7 with new panel 7D). Importantly, both effects are rectified by the reduction-of-function Mtorchino mutation (Figure 11A-D with new panel 11A), demonstrating a central role for mTOR.

Thanks to the constructive criticism we received, we believe that the changes underlined in our revised Title, Abstract, Introduction, Results, and Discussion help to explain our data more clearly, and offer testable hypotheses for the underlying mechanisms.

2) Although the mutant mice showed significant reduction of the ERK signaling, the authors concluded that there were no abnormalities in CD4SP thymocytes, regulatory T cells, and clonal deletion. The authors need to examine whether their functions, such as suppressive activity of regulatory T cells, are normal.

We have clarified in text in the Discussion that although we do not observe any deficit in frequency of Foxp3+ Tregs, we have not tested if these Tregs have normal suppressive activity. We do conclude that should any deficit in Treg suppressive activity exist, it does not account for the increased CD44 on naïve CD4s nor the increased formation of CD4 effectors, because those abnormalities are cell autonomous and not corrected in mixed chimeras where there are large numbers of wildtype Tregs. The following text has been added:

“A putative defect in regulatory T cells cannot explain the increased CD44 expression on naïve Rasgrp1Anaef CD4+ cells, because in mixed chimeras bearing many wild-type Foxp3+ CD4 cells, CD44 expression was still increased on Rasgrp1Anaef but not on co-resident wild-type CD62L+ FOXP3 CD4+ splenocytes.”

3) In Figure 5A, the reduced levels of P-T184 in the Rasgrp1Anaef thymocytes may be due to the reduced levels of Rasgrp1Anaef protein and to the poor stability of Rasgrp1Anaef following TCR stimulation. To dismiss this possibility, the blot needs to be immune-blotted with an anti-Rasgrp1 antibody (as shown in Figure 2E). In addition, the Rasgrp1Anaef mutation could be more similar to the Rasgrp1null mutation compared with the Rasgrp1+/- mutation. To compare functional differences, it is recommended to include the Rasgrp1null mutation in Figure 5.

We agree with this point. Indeed the total Rasgrp1 expression levels that accompany Figure 5A (and 5B) were shown in supplemental figure S4c of the original manuscript, and these immunoblots demonstrate that the loss of T184 phosphorylation is not due to the decreased expression of total Rasgrp1. For the sake of clarity we have now eliminated that figure and included all the controls in Figures 5A and 5B.

Regarding direct comparisons with Rasgrp1 null thymocytes, TCR-induced ERK activation is severely impaired in Rasgrp1 null thymocytes and this has been published (e.g., Dower et al., Nature Immunology). Since Rasgrp1 deficiency results in a severe block in thymocyte development, Rasgrp1 null thymi consist almost entirely of CD4CD8 double positive thymocytes with low levels of TCR expression. There are virtually no CD4 or CD8 single positive cells that are more mature and express higher levels of surface TCR. Very differently, WT of Rasgrp1Anaef thymi contain all normal thymocyte populations. Functional comparisons of TCR-induced PLCgamma and ERK activation in Rasgrp1Anaef (or WT) thymocytes with the signals in Rasgrp1null thymocytes are not very informative (and perhaps even confusing for the reader) since these comparisons involve not only different developmental thymocyte subsets but also have the caveat of comparing cells with different levels of TCR expression, which is known to affect the efficiency of TCR stimulation.

4) In Figure 10, the data suggesting that “substantial” levels of P-S6 are found in freshly isolated lymph nodes need to be shown.

We thank the reviewers for this suggestion – these data have now been added as new Figure 10B, which shows immunoblots of extracts from lymph node cells before or after a 30 minute PBS rest, and probing for total phospho-tyrosine, pZap70, pErk, and pS6. The blots clearly demonstrate that phosphorylated signaling molecules, including S6, are readily detectable in freshly isolated samples and that these levels decrease upon brief resting in PBS. We also moved a panel from the supplement to become new Figure 10C, which shows tonic S6 signals in purified CD4 T cells.

5) The conclusion that tonic TCR signal results in basal mTOR-S6 needs to be further substantiated since it is based on the aggregation of disparate data from the Germain laboratory and from the authors’ laboratory.

We thank the reviewers for this important query. We performed an experiment to test the role of self- peptide/MHCII recognition, and found that the elevated CD44 levels on naïve CD4 T cells from the Anaef mice is retained when cells are transferred into MHCII knockout mice These results are presented in new Figure 10E. Therefore we inserted the following text into the Results:

“To examine if self-peptide/MHCII recognition plays a role in the increased CD44 expression on Rasgrp1Anaef naïve CD4+ T cells, we adoptively transferred a mixture of wild-type and Rasgrp1Anaef splenocytes into wild-type or MHCII(H2-Aa)–deficient recipient mice…”

6) To identify the mutated gene, the authors narrowed down the region where the mutation located and picked up Rasgrp1 because it was an only gene with an immune function within the located region. As some phenotypes were different between Rasgap1null and Rasgap1Anaef mutation, is there a possibility that another mutation locating on the adjacent region is involved in the observed phenotypes? In this context, if the progeny is generated from Rasgrp1Anaef mice tha are ANA negative (Figure 1D), are they ANA positive?

The fact that RasGRP1-deficient Jurkat T cells reconstituted with a Rasgrp1Anaef allele and Rasgrp1Anaef thymocytes demonstrated very similar Ras-ERK responses upon in vitro stimulation (Figsures 4 and 5) already argued against another mutation causing the phenotype in Rasgrp1Anaef mice. However, to be certain there was no other mutation closely linked to the Rasgrp1Anaef mutation, whole-exome capture, sequencing and computational analysis were performed on DNA from an affected mouse. This identified the Rasgrp1Anaef mutation as the only novel homozygous single-nucleotide variant on chromosome 2 between 104.9 Mb and 145.7 Mb, a wide interval in which each boundary was marked by two affected F2 mice that inherited recombined chromosomes (Figure 1–figure supplement 1). We added the following text:

“Whole-exome capture, sequencing and computational analysis of DNA from an affected mouse (Andrews et al., 2012) identified this mutation as the only novel single-nucleotide variant within the interval of interest on chromosome 2 (data not shown).”

7) It might be interesting if the authors quickly review papers that have genetically deleted mTOR signaling in T cells to see if there might be differences in CD44 expression in those mice that might not have been emphasized in the original papers.

We thank the reviewers for this suggestion and have added further details of the connection between CD44 levels and mTOR.

“This conclusion is reinforced by supplementary data from two studies: mice with a neo-insertion in an Mtor intron that decreases Mtor mRNA approximately 70% show a similar selective lowering of CD44 on CD4+ T cells (Zhang et al., 2011) and CD44 levels are also reduced on naïve, CD62L-positive T cells that are deficient for the mTOR activator Rheb or deficient for the mTOR-interacting protein Rictor (Delgoffe et al., 2011)…”


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