ARTICLE RRAS2 shapes the TCR repertoire by setting the threshold for negative selection Ana Martı́nez-Riaño1, Elena R. Bovolenta1, Viola L. Boccasavia1, Julia Ponomarenko2,3, David Abia4, Clara L. Oeste1, Manuel Fresno1, Hisse M. van Santen1, and Balbino Alarcon1 Signal strength controls the outcome of αβ T cell selection in the thymus, resulting in death if the affinity of the rearranged TCR is below the threshold for positive selection, or if the affinity of the TCR is above the threshold for negative selection. Here we show that deletion of the GTPase RRAS2 results in exacerbated negative selection and above-normal expression of positive selection markers. Furthermore, Rras2−/− mice are resistant to autoimmunity both in a model of inflammatory bowel disease (IBD) and in a model of myelin oligodendrocyte glycoprotein (MOG)–induced experimental autoimmune encephalomyelitis (EAE). We show that MOG-specific T cells in Rras2−/− mice have reduced affinity for MOG/I-Ab tetramers, suggesting that enhanced negative selection leads to selection of TCRs with lower affinity for the self-MOG peptide. An analysis of the TCR repertoire shows alterations that mostly affect the TCRα variable (TRAV) locus with specific VJ combinations and CDR3α sequences that are absent in Rras2−/− mice, suggesting their involvement in autoimmunity. Introduction T cell development in the thymus is an active process that im- thymocytes and controls their fate (death, survival, and differ- plies different intracellular signaling events regulating cell dif- entiation; Starr et al., 2003; Gascoigne et al., 2016). ferentiation, proliferation, and survival. This process generates Engagement of the TCR with cognate pMHC promotes a an anticipatory peripheral T cell repertoire able to promote conformational change and the initiation of downstream sig- a proper adaptive defense to future antigens derived from naling cascades, beginning with the recruitment of Nck and pathogens. Thymic development follows well-defined matura- phosphorylation of CD3 immunoreceptor tyrosine activation tion steps according to the expression of CD4 and CD8 cor- motifs (ITAMs) by the Src-kinase, Lck. ITAM phosphorylation eceptors: CD4−CD8− (double negative [DN]), CD4+CD8+ (double generates docking sites for the Syk-tyrosine kinase ZAP-70, positive [DP]), and CD4+ or CD8+ (single positive [SP]; SP4 or which in turn phosphorylates the adaptor LAT and promotes the SP8). At the DN stage, the expression of a correctly rearranged generation of the early TCR scaffold complex together with TCRβ chain together with the invariant pTα chain allows thy- SLP76 and PLCγ1, activating multiple biochemical pathways mocytes to mature to the DP stage and rearrange the TCRα chain (Cantrell, 2015; Alcover et al., 2018). In vivo studies have shown to express a mature α/β TCR (Falk et al., 2001). To promote only that thymocyte selection is dependent on the activation of early the survival of functional self-tolerant T cells, DP thymocytes are TCR signaling molecules, such as the kinases and adaptors Lck, subjected to a stringent selection process based on the affinity of ZAP70, Nck, LAT, and SLP76 (Zamoyska et al., 2003; Singer their randomly recombined TCR for self-peptides presented by et al., 2008; Borroto et al., 2013). In addition to those TCR sig- MHC molecules (self-pMHC). Thymocytes with TCRs that fail to nal transducers that play a role at all stages of a T cell’s life, new interact, and those with TCRs that interact with too much af- molecular players are being discovered, such as Themis and finity with self-pMHCs, enter into an apoptotic process called Tespa1, which regulate TCR signaling during negative and pos- “death by neglect” or “negative selection,” respectively. Only itive selection (Wang et al., 2012; Gascoigne et al., 2016; Choi those thymocytes with TCRs that interact with low but sufficient et al., 2017). affinity are “positively selected” and progress toward SP4 or SP8 Activation of the TCR downstream pathways PI3K-Akt and stages. Hence, TCR signaling is essential during development of ERK via Ras proteins is of great importance during thymocyte ............................................................................................................................................................................. 1Departamento de Biologı́a Celular e Inmunologı́a, Centro Biologı́a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı́ficas, Universidad Autónoma de Madrid, Madrid, Spain; 2Centre for Genomic Regulation, The Barcelona Institute of Science and Technology, Barcelona, Spain; 3Universitat Pompeu Fabra, Barcelona, Spain; 4Servicio de Bioinformática, Centro Biologı́a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı́ficas, Universidad Autónoma de Madrid, Madrid, Spain. Correspondence to Balbino Alarcon: balarcon@cbm.csic.es; Hisse M. van Santen: hvansanten@cbm.csic.es. © 2019 Martı́nez-Riaño et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press https://doi.org/10.1084/jem.20181959 2427 J. Exp. Med. 2019 Vol. 216 No. 10 2427–2447 development. Thus, PI3K-Akt has been described to control pre- presented a small, but significant, reduction in the percentage of TCR-dependent differentiation, positive and negative selection DP (CD4+CD8+) thymocytes, accompanied by a small increase in signals, CD4/CD8 ratio, differentiation, and thymic exit (Na the percentage of CD4SP and CD8SP (Fig. 1 B). The effects on the et al., 2003; Rodrı́guez-Borlado et al., 2003; Barbee and relative distribution of thymic populations were accompanied Alberola-Ila, 2005; Fayard et al., 2010). Likewise, Ras-ERK sig- by a slight reduction of TCR expression in Rras2−/− mice com- nals have been shown to be required for pre-TCR signaling, as pared with WT in the DP, CD4SP, and CD8SP populations (Fig. 1 well as to determine positive or negative selection, depending on C). This reduction of TCR expression was concomitant with a the kinetics of ERK activation (Fischer et al., 2005; Daniels et al., small but significant reduction of TCR and CD4 or CD8 cor- 2006). The Ras family of small GTPases consists of 39 genes in eceptor expression in mature T cells of peripheral lymphoid humans (Rojas et al., 2012). Although some of their members are organs (Fig. S1 A). The reduced expression of the TCR and cor- frequently mutated in human cancer, their role in physiological eceptors may be indicative of a defect in positive or negative processes is not as well defined. Using germline knockout mouse selection at the DP stage in Rras2−/− mice. Another piece of in- lines of classical RAS subfamily members Nras and Hras, it was formation that indicated a defect in positive or negative selec- shown that these genes mediate neither positive nor negative tion at the DP stage was that the number of DP thymocytes selection (Iborra et al., 2011). In contrast, the implication of negative or with low TCR (or CD3) expression was not smaller in several guanosine nucleotide exchange factor proteins such as Rras2−/− mice than in WT controls (Fig. 1 D). In fact, the number RasGRP and Sos in thymocyte selection processes is remarkable, of DP CD3low thymocytes was a bit higher, in line with the suggesting that Ras GTPases are indeed involved (Dower et al., general tendency to have bigger thymuses in the absence of 2000; Iborra et al., 2011; Kortum et al., 2013). In our previous RRAS2 (Fig. 1 A). The result was very different when the number work, we described that RRAS2, a small GTPase of the RRAS of DP thymocytes positive for CD3 was analyzed; Rras2−/− mice subfamily, interacts with ITAMs of the TCR and BCR and that it have reduced number of DP positive thymocytes once they ex- plays a fundamental role in the survival and homeostatic pro- press the TCR (Fig. 1 D). This result shows that defects in Rras2−/− liferation of mature T and B cells, presumably through the ac- mice during thymic selection occur after the TCR is expressed. tivation of the PI3K-Akt pathway (Delgado et al., 2009; We did not detect major alterations in the percentage of Martı́nez-Martı́n et al., 2009). More recently, we have shown natural regulatory T cells (nT reg cells) within the CD4SP pop- that RRAS2 has a nonredundant, B cell–intrinsic essential role ulation in the thymus. This is to be expected given that nT reg during the germinal center response by controlling B cell me- cell differentiation is believed to occur for thymocytes with TCR tabolism (Mendoza et al., 2018). affinities for self-pMHC that lie between the affinities that drive In the present study, we describe the relevance of RRAS2 positive and negative selection (Hsieh et al., 2012; Klein et al., during T cell development. We found that Rras2─/─ mice have 2014). Instead, we did detect a small reduction of the percentage reduced DP thymocyte numbers and show an activated thymo- of nT reg cells in spleen, but not in lymph nodes, of Rras2−/− cyte phenotype with increased up-regulation of Nur77 and Bim compared with WT mice (Fig. S1 B). pro-apoptotic molecules. Moreover, the absence of RRAS2 in To determine if RRAS2 deficiency leads to defective selection DP OT-I TCR transgenic thymocytes makes them more sensitive in the thymus, we crossed Rras2−/− mice and their WT coun- to apoptosis induced by OVA antigen-variant peptides of low terparts with mice transgenic for the AND TCR (ANDTg). The affinity, indicating an enhanced negative selection process. AND TCR is specific for a moth cytochrome c peptide presented Consequently, Rras2─/─ mice have an altered peripheral TCR by I-Ek (Kaye et al., 1989). The number of ANDTg thymocytes in repertoire and show less susceptibility to undergo autoimmune mice homozygous for the I-Ek allele is reduced in comparison disorders, such as inflammatory bowel disease (IBD) or experi- with I-Ek heterozygous mice (Kaye et al., 1992). Importantly, this mental autoimmune encephalomyelitis (EAE). We hypothesize suggests that the I-Ek allele induces a certain extent of negative that RRAS2 could be an important molecule in the TCR signaling selection, possibly because of a higher affinity of the ANDTg cascade, setting the threshold for negative selection in thymo- receptor for this allele compared with the also positively se- cytes by activating the pro-survival PI3K-Akt pathway. lecting I-Ab allele (Kaye et al., 1992). To determine whether RRAS2 affected negative selection, we backcrossed ANDTg Rras2−/− mice in mixed k/b background with C57BL/6 mice for Results eight generations to create a line of mice homozygous for the H-2b Reduced numbers of DP thymocytes in Rras2−/− mice haplotype (b/b) or bearing one H-2k haplotype (k/b). ANDTg To investigate the role of RRAS2 during T cell maturation in the WT DP thymocytes are selected toward CD4SP mature thymo- thymus, we first studied the distribution of thymocytes within cytes in both the k/b and b/b haplotype (Fig. 1 E). Compared with the four major thymic populations in Rras2−/− mice and WT WT, mice deficient in RRAS2 in a k/b background had a lower (Rras2+/+ WT) controls. We found that thymuses of Rras2−/− mice percentage of DP thymocytes and slightly higher percentage of were slightly bigger than those of their WT counterparts and had CD4SP (Fig. 1 E). The analysis of the number of thymocytes, increased numbers of thymocytes of the DN (CD4–CD8–), CD4SP however, showed a strong and significant reduction of the DP (CD4+CD8–), and CD8SP (CD4–CD8+) populations, although those and the CD4SP populations (Fig. 1 F). By contrast, there were no differences were only significant for CD4SP cells (Fig. 1 A). significant differences in the distribution or size of thymic However, analysis of RRAS2 deficiency in terms of the relative populations between WT and Rras2−/− mice in the absence of the distribution into the four populations showed that Rras2−/− mice I-Ek haplotype (b/b background; Fig. 1, E and F). AND TCR Martı́nez-Riaño et al. Journal of Experimental Medicine 2428 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Martı́nez-Riaño et al. Journal of Experimental Medicine 2429 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 1. Reduced percentage of DP thymocytes in Rras2-deficient mice. (A) Flow cytometry analysis of WT and Rras2-deficient thymocytes in 6-wk-old C57BL/6 mice according to the expression of CD4 and CD8α markers. Bar plots to the right represent the number of CD4ˉCD8ˉ (DN), CD4+CD8+ (DP), CD4+CD8ˉ (CD4SP), CD4ˉCD8+ (CD8SP), and total thymocytes in both genotypes (n = 8 mice per group). Blue circles, Rras2+/+; red squares, Rras2−/−. (B) Percentages of DN, DP, SP4, and DP8 thymocytes in mice analyzed as in A. n = 11 mice per group. (C) Flow cytometry analysis of CD3 expression in the indicated thymocyte populations. An overlay plot of Rras2+/+ (continuous line) and Rras2−/− (broken lines) is shown on the left, and quantification of CD3 expression is shown in the bar blots on the right. n = 4 mice per group. MFI, mean fluorescence intensity. (D) Number of DP thymocytes according to the expression of CD3. Two-color plots show CD3 expression versus DP thymocyte size. Bar plots below represent the number of DP thymocytes expressing or not expressing CD3 for both genotypes Rras2+/+ (blue circles) and Rras2−/− (red squares). n = 9 mice per group. FS, forward scatter. (E) Flow cytometry analysis of WT and Rras2-deficient thymocytes in ANDTg mice in a pure b/b background and in a mixed k/b background after gating within the transgenic Vβ3high thymocytes. Bar plots to the right represent the percentage of thymocyte subpopulations for both genotypes. Blue circles, Rras2+/+; red squares, Rras2−/−. n = 4–7 mice per group. (F) Number of DN, DP, SP4, and DP8 thymocytes in AND TCR mice in a pure b/b background and in a mixed k/b background. Blue circles, Rras2+/+; red squares, Rras2−/−. n = 4–6 mice per group. Quantitative data in all panels are means ± SEM. *, P < 0.05; **, P < 0.005; ****, P < 0.0005; n.s., not significant (unpaired two-tailed Student’s t test). All phenotyping experiments in this figure were repeated at least three times. expression was not significantly different in DP and CD4SP beyond the DN stage in both TCR transgenic and nontransgenic thymocytes of WT or Rras2−/− mice in a k/b background (Fig. S1 mice bearing a polyclonal TCR repertoire. D). Therefore, the reduced number of DP and CD4SP thymocytes in the presence of the I-Ek haplotype suggests that the absence of Enhanced negative selection in Rras2−/− mice RRAS2 enhances negative selection. The differential effect of RRAS2 deficiency on selection of ANDTg To investigate if the effect of RRAS2 deficiency on thymic thymocytes in the presence and absence of I-Ek (Fig. 1, E and F) selection was T cell–intrinsic, we adoptively transferred bone suggested that reduced DP thymocyte number was due to en- marrow precursors from ANDTg Rras2−/− or WT mice of the hanced negative selection. To further investigate if RRAS2 de- b/b background into lethally irradiated WT nontransgenic k/b ficiency enhanced negative selection or was, in contrast, the recipient mice. Thymuses of reconstituted mice were ana- consequence of deficient positive selection, we analyzed the lyzed for major subset distribution within the donor-derived, expression of positive and negative selection markers in DP, ANDTg-specific cells (Vβ3high, H-2k–negative). The compari- transitional, and CD4SP ANDTg thymocytes in the k/b back- son between donor cells of WT and Rras2−/− genotypes showed ground. DP thymocytes that receive a TCR signal up-regulate a strong reduction in the number of DP and CD4SP thymocytes CD69 and CD5, which are considered markers of TCR signaling in the absence of RRAS2 (Fig. 2 A). This result shows that the strength (Bendelac et al., 1992; Brändle et al., 1994; Azzam et al., effect of RRAS2 deficiency on thymic selection is indeed 1998). In fact, CD69 expression correlates with the nature of T cell–intrinsic. positive or negative selecting peptides and is higher in the latter At this point, we examined whether the relative reduction of (Merkenschlager et al., 1997). When comparing the expression DP thymocytes detected in Rras2−/− mice with a polyclonal TCR of CD5 and CD69 in DP and CD4SP thymocytes of WT and repertoire (Fig. 1 A) was also T cell intrinsic. To enhance our Rras2−/−mice, we found that RRAS2 deficiency did not decrease possibility of observing the potential effect of RRAS2 deficiency, the expression of those markers. Rather, it had the opposite we performed this experiment under competitive conditions. effect, such that CD5 and CD69 were significantly more up- Donor bone marrow cells of WT or Rras2−/− genotype bearing the regulated than in WT thymocytes (Fig. 3 A). This increased CD45.2 allele were mixed with an equal proportion (50:50) of up-regulation of CD5 and CD69 markers suggests that ANDTg donor WT bone marrow cells bearing the CD45.1 allele. The Rras2−/− thymocytes are not deficient in positive selection. To mixture was subsequently inoculated into lethally irradiated test if the same effect held true for non-TCR transgenic thy- CD45.1+ WT recipient mice (Fig. 2 B). 2 mo after bone marrow mocytes, we studied the expression of CD5 and CD69 in thymic reconstitution, the expression of the CD45.2 allele within the Rras2−/− and WT populations under the competitive conditions myeloid CD11b+ compartment in the spleen was close to 50%, described for the experiments in Fig. 2. We found that both regardless of the Rras2 genotype (Fig. 2 C). The analysis of markers were significantly more up-regulated in Rras2−/− DP thymic populations showed, by contrast, a very different dis- thymocytes than in their WT counterparts and that Rras2−/− and tribution. The percentage of thymocytes bearing the CD45.2 WT SP thymocytes showed no differences in expression level allele within the DP, CD4SP, and CD8SP subsets was strongly (Fig. 3 B). These data suggest that positive selection was not reduced in Rras2−/− donor thymocytes, compared with their WT impaired in the absence of RRAS2 and that Rras2−/− DP thymo- controls (Fig. 2, D and E). The percentage of CD45.2+ thymocytes cytes actually received stronger signals via their TCR. within the DN subset did not differ significantly between the Another marker of TCR signaling strength is the orphan two genotypes (Fig. 2 E). Furthermore, the distribution of DN nuclear hormone receptor Nur77, which is considered a marker thymocytes into the four major subpopulations defined by the of negative selection in vivo (Moran et al., 2011). Nur77 ex- expression of CD44 and CD25 markers was not significantly pression was up-regulated in ANDTg DP (CD8high in Fig. 3 C) and different between WT and Rras2−/− mice in the bone marrow CD4SP thymocytes going through intermediate CD8low stages reconstitution experiment (Fig. S1 E), either in nontransgenic (CD8low, Fig. 3 C) in Rras2−/− mice compared with WT. To de- mice (Fig. S1 F) or in ANDTg mice (Fig. S1 G). These data indicate termine if additional negative selection markers were up- that RRAS2 deficiency induces a defect in thymic selection regulated in the absence of RRAS2 in non-TCR transgenic Martı́nez-Riaño et al. Journal of Experimental Medicine 2430 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 2. Reduced numbers of DP thymocytes in Rras2−/− mice result from a T cell–intrinsic defect. (A) WT adult mice of mixed k/b background were lethally irradiated and reconstituted with bone marrow cells from either Rras2+/+ or Rras2−/− AND TCR transgenic mice of b/b background. 2 mo after re- constitution, thymocyte count and DP and CD4SP subset distribution were analyzed within the donor H-2k negative Vβ3+ (AND b/b) population. Quantitative data are means ± SEM (n = 3). *, P < 0.05; **, P < 0.005 (unpaired two-tailed Student’s t test). This experiment was repeated twice. (B) WT adult mice of b/b background expressing the CD45.1 allele were lethally irradiated and reconstituted with an equal mixture of bone marrow cells from WT CD45.1+ mice and Martı́nez-Riaño et al. Journal of Experimental Medicine 2431 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 either Rras2+/+ or Rras2−/− mice expressing the CD45.2 allele. (C) 2 mo after reconstitution, chimerism was analyzed by measuring the distribution of the CD45.2 allele within myeloid CD11b+ spleen cells. (D) The effect of RRAS2 deficiency on the distribution of thymocyte subpopulations was analyzed according to the expression of the CD45.2 allele. An example of CD45.2 expression within the DP thymocyte population of chimeric mice is shown in the plots below. (E) Relative contribution of the donor CD45.2+ cells to the different thymocyte populations. Data points from two different experiments are integrated by normalizing the percentage of CD45.2+ cells to those of the mean values in the WT controls within each experiment. Quantitative data in all panels are means ± SEM (n = 5 mice per group). ****, P < 0.00005; ***, P < 0.0005; (unpaired two-tailed Student’s t test). The experiment of B–E was repeated twice. n.s., not significant. thymocytes, we performed a quantitative RT-PCR (RT-qPCR) (Teixeiro and Daniels, 2010). Thus, negative selection correlates analysis of gene expression on FACS-sorted DP thymocytes us- with an intense but brief phosphorylation of ERK, whereas ing a panel of genes previously characterized as differentially positive selection correlates with weaker but more sustained regulated during positive or negative selection (Mingueneau ERK phosphorylation. On the other hand, although classic Ras et al., 2013). The comparison between Rras2−/− and WT geno- GTPases activate the Raf-ERK pathway, RRAS2 is rather an types showed a significant transcriptional up-regulation of activator of the PI3K-Akt pathway. We therefore investigated genes associated with negative selection, whereas expression of induction of both pathways after a short-term stimulation with genes associated with positive selection was not significantly anti-CD3, a stimulus likely mimicking negative selection. We altered in the absence of RRAS2 (Fig. 3 D). Altogether, the results first analyzed the induction of phosphorylation of ERK and Akt shown in Fig. 3 indicate that positive selection is not deficient in by Western blotting of post-nuclear lysates of total thymocytes Rras2−/− mice and that stronger negative selection occurs ac- stimulated with anti-CD3 in vitro. We found that dual ERK cording to the specific marker expression. phosphorylation by MEK-1 at Thr202 and Tyr204 peaked at Another model to study the effect of RRAS2 deficiency on 3 min of stimulation with similar kinetics in thymocytes from positive versus negative selection is that of OT1 TCR-transgenic WT and Rras2−/− mice (Fig. 5 A). However, phosphorylation of mice. The OT1 TCR recognizes a peptide derived from OVA Akt at residue Ser473 by mTORC2 and phosphorylation of S6 by (OVAp) presented by H2-Kb and drives differentiation of OT1 p70S6K, which is downstream of Akt, in residue Ser240 were T cells into the CD8+ lineage. A phenotypic analysis of adult reduced at all time points in the absence of RRAS2 compared Rras2−/− and WT mice showed that the former had relatively with the WT. These results suggested that ERK phosphorylation fewer DP thymocytes (CD4highCD8high, Fig. 4 A) and more is not affected by RRAS2 deficiency, whereas activation of the transitional CD4lowCD8low and CD4+CD8low thymocytes. Unlike PI3K-Akt pathway is strongly inhibited. in mice with a polyclonal TCR repertoire (Fig. S1), OT1 CD8 We also examined if RRAS2 deficiency altered the phospho- T cells showed no differences in the expression of CD8β in pe- rylation of MKK6 (p38), since higher activation of this MAP ripheral T cells from Rras2−/− mice compared with WT (Fig. 4 B). kinase has been correlated with stronger negative selection A sharp affinity threshold between positively selecting and (Sugawara et al., 1998). We detected reduced phosphorylation of negatively selecting peptides for the OT1 TCR has been defined p38 in thymocytes of Rras2−/− mice (Fig. 5 A), suggesting that based on the induction of CD69 expression (Daniels et al., 2006). enhanced negative selection in RRAS2-deficient mice is not due We incubated total thymocytes from Rras2−/− and WT OT1Tg to increased p38 activity. mice for 24 h with splenic antigen-presenting cells loaded with Since Western blotting was performed on total thymocyte OVAp variants with characterized affinity for the OT1 TCR. In lysates, we also measured ERK and Akt phosphorylation in this system, the Q4H7 peptide variant is positive selecting, thymic subsets by phospho-flow cytometry to study their reg- whereas the Q4R7 peptide is just above the threshold of affinity ulation in thymic subpopulations. Induction of Akt phospho- necessary for negative selection (Daniels et al., 2006). We found rylation by stimulation with anti-CD3 was not detectable in DP, that all OVAp derivatives induced more CD69 and annexin V CD4SP, or CD8SP thymocytes of Rras2−/− mice, whereas clear expression (a marker of apoptosis) in RRAS2-deficient DP induction was observed in WT controls (Fig. 5 B). The effect of thymocytes than in their WT counterparts regardless of their RRAS2 deficiency on ERK phosphorylation was, however, more concentrations and affinities (Fig. 4 C). These results are complex: whereas ERK phosphorylation peaked between in agreement with the observation that induction of the TCR 3–5 min in RRAS2-deficient DP and SP thymocytes as in the WT engagement marker CD69 in Rras2−/− DP thymocytes is not weaker controls, ERK phosphorylation was less sustained after 5 min but rather stronger than in WT DP ones and that induction of of stimulation (Fig. 5 B). In summary, the Western blot and apoptosis is consistently stronger. These results suggest that phospho-flow data indicate that thymocytes from Rras2−/− mice RRAS2 deficiency does not lead to defective TCR signaling in are deficient in the activation of the PI3K-Akt pathway upon response to low-affinity antigens in OT1 mice and reinforce the stimulation with anti-CD3 and that ERK phosphorylation might idea that RRAS2 deficiency enhances negative selection. be less sustained than in WT thymocytes. Defective PI3K-Akt pathway activation in thymocytes from Resistance to autoimmunity in Rras2−/− mice Rras2−/− mice A consequence of enhanced negative selection in the thymus Differences in the intensity and kinetics of activation of the Raf- should be reduced autoreactivity of the mature T cells exiting ERK pathway after TCR triggering have been correlated with the thymus and a reduction in the intensity or the propensity to the effect of positive-selecting versus negative-selecting ligands develop autoimmune diseases in which T cells are a major Martı́nez-Riaño et al. Journal of Experimental Medicine 2432 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 3. Enhanced expression of positive selection and negative selection markers in thymocytes from Rras2−/− mice. (A) Thymocytes from AND TCR transgenic mice in mixed k/b background were analyzed for expression of positive selection markers CD5 and CD69 within the DP and CD4SP subsets. Bar plots show quantitative data from a total of three experiments. Percentage and mean fluorescence intensity (MFI) values are represented normalized to those of the mean values in the WT controls within each experiment. Mean percentage and MFI in WT thymocytes are set to 1. Quantitative data are means ± SEM (n = 7–12 mice per group). *, P < 0.05; **, P < 0.005 (unpaired two-tailed Student’s t test). This experiment was repeated three times. (B) Expression of positive selection markers CD5 and CD69 in the thymic subsets of CD45.2+ thymocytes from the bone marrow competition experiments of Fig. 2, B–E. An example of Martı́nez-Riaño et al. Journal of Experimental Medicine 2433 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 the gating strategy is shown for the DP population. Quantitative MFI data for the entire DP, CD4SP, and CD8SP populations are means ± SEM (n = 5 mice per group). *, P < 0.05 (unpaired two-tailed Student’s t test). This experiment was repeated twice. (C) Thymocytes from AND TCR transgenic mice in mixed k/b background were analyzed for expression of the negative selection marker Nur77 within CD4+ subsets, as indicated. Bar plots show the normalized expression of Nur77 in the CD8high (DP) and CD8low (transitional and CD4SP) subpopulations in a combination of three experiments. MFI values are represented nor- malized to those of the mean values in the WT controls within each experiment. Quantitative data are means ± SEM (n = 8–9 mice per group). **, P < 0.005 (unpaired two-tailed Student’s t test). This experiment was repeated three times. (D) RT-qPCR analysis of gene expression in sorted DP thymocytes from Rras2+/+ and Rras2−/− mice. Genes are grouped according to their differential expression during positive and negative selection (Mingueneau et al., 2013). An interleaved box and whiskers plot showing all data points and the maximum and minimum has been chosen for data representation. Values are normalized to Rras2+/+. *, P < 0.05. An unpaired two-tailed Student’s t test was used to compare significance of individual genes; a two-way ANOVA test was used to compare gene sets. This experiment was repeated four times. n.s., not significant. driver. To test this hypothesis, we first selected a model of IBD, resistance to EAE of Rras2−/− mice is at least in part T cell in which CD4+ T cells depleted of nT reg cells are adoptively intrinsic. transferred into lymphopenic mice. The homeostatic expansion We next searched for the presence of MOG-responsive pro- of the transferred T cells is supposed to benefit the expansion of inflammatory T cells in lymphoid organs of immunized mice. autoreactive T cells, which infiltrate the intestinal mucosa and We found a reduced percentage of IFNγ- and IL-17A–producing cause symptoms similar to those observed in Crohn’s disease CD4+ T cells in draining lymph nodes of Rras2−/− mice compared and ulcerative colitis. The adoptive transfer of CD4+CD25− T cells with WT (Fig. 7 C), suggesting that a weaker reactivity with from WT donors to RAG1−/− lymphopenic mice caused a pro- MOG leads to less generation of pro-inflammatory effector CD4 gressive loss of body weight that was not detected when trans- T cells in RRAS2-deficient mice. By contrast, we did not detect ferred T cells originated from Rras2−/− mice (Fig. 6 A). Body significant differences in the percentage of Foxp3+CD25+CD4+ T weight loss in mice receiving WT T cells was accompanied by an reg cells, suggesting that resistance to EAE is not due to a higher increase in the length and weight of the colon, as well as the abundance of T reg cells (Fig. 7 C). colon weight/length ratio, compared with those of the RAG1−/− The mice in Fig. 7 A were sacrificed at day 30, and the control mice not inoculated with T cells (Fig. 6 B). In contrast, presence of MOG-reactive CD4+ T cells was evaluated in spleens RAG1−/− mice that received T cells from Rras2−/− mice were in- by flow cytometry, gating on the CD4+TCRβ+ T cell population distinguishable from noninoculated mice. The absence of IBD bearing the CD44 marker of activation. Staining with different symptoms in mice transferred with RRAS2-deficient T cells was dilutions of an I-Ab tetramer loaded with the immunizing MOG not due to a defective expansion of the cells, because CD4+ T cell peptide allowed us to detect 8% MOG tetramer+ CD4+CD44+ numbers detected in spleens of adoptively transferred mice T cells in WT mice and 6% in Rras2−/− mice (Fig. S2 A). The were not significantly different from those of mice transferred poorer expansion of MOG tetramer+ CD4 T cells in Rras2−/− mice with WT cells (Fig. 6 C). Although the number of total CD4+ could result from an underlying defect of RRAS2 deficient ma- T cells was not significantly different, the presence of pro- ture T cells in responding to TCR triggering. However, we found inflammatory effector Th cells producing IFNγ or IL-17A in that the response of Rras2−/−CD4+ and CD8+ T cells to anti-CD3 spleen was significantly reduced in mice that received RRAS2- stimulation in terms of CD69 expression and cytokine produc- deficient versus WT T cells (Fig. 6 C). By contrast, no differences tion (IL-2 and IFNγ) were unaffected (Fig. S3, A and B). The were observed for the Foxp3+CD25+–induced T reg cell popula- proliferative response to anti-CD3 plus anti-CD28 stimulation tion. These results indicate that conventional CD4+ T cells se- was not impaired in Rras2−/− T cells; in fact, RRAS2-deficient lected in the absence of RRAS2 are unable to promote IBD. CD4 T cells proliferated faster than their WT counterparts We used a second model of autoimmunity, EAE, in which (Fig. S3 C). These data indicate that the response to polyclonal tolerance to self-antigens is broken by immunization with a self- stimulation of mature T cells is not defective in the absence of peptide in the presence of a strong adjuvant. This model allows RRAS2. Furthermore, the proliferative response of OT1 CD8+ detection of T cells reactive with the self-peptide used for im- T cells to stimulation with its agonist peptide in vivo was in- munization. EAE is induced upon immunization of female distinguishable from those of WT cells (Fig. S3 D). We therefore C57BL/6 mice with a peptide derived from myelin oligoden- reasoned that the dissimilar percentages of MOG tetramer+ CD4 drocyte glycoprotein (MOG peptide). Immunization of WT mice T cells in the EAE experiment in vivo were due to a different led to a progressive loss of neurological capabilities that led to a responsiveness derived from an exacerbated negative selection complete paralysis of the hind limbs (Fig. 7 A). Immunization of in the thymus. Along these lines, staining with the MOG tetra- Rras2−/− mice led to a milder disease with faster recovery from mer was weaker in Rras2−/− mice at all tetramer dilutions in symptoms and less body weight loss than immunized WT mice spite of the fact that TCR expression was identical to WT MOG (Fig. 7 A). We next tested if the milder form of EAE in Rras2−/− tetramer+ T cells, as shown by co-staining with an anti-TCRβ mice compared with WT was T cell intrinsic. To this aim, we antibody (Fig. S2 A). Interestingly, we did not detect reduced reconstituted lethally irradiated WT mice with bone marrow of reactivity of Rras2−/− mice to immunization with a nonself OVAp either WT or Rras2−/− mice and immunized with MOG 2 mo after compared with their WT counterparts (Fig. S2 B), suggesting reconstitution. RRAS2-deficient mice developed EAE symptoms that the response to strong, foreign antigens is not diminished in at a significantly lower rate than WT mice and began to lose the thymus. The reduction of the percentage of MOG tetramer+ weight with a delay of 4 d (Fig. 7 B). These data indicate that the CD4+ T cells (Fig. S2 A) in Rras2−/− mice data suggested a weaker Martı́nez-Riaño et al. Journal of Experimental Medicine 2434 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 4. Enhanced activation of Rras2-deficient DP thymocytes in response to low- and high-affinity TCR ligands. (A) Rras2+/+ or Rras2−/− OT1 TCR transgenic mice were analyzed for the distribution of thymocytes among DP (CD4highCD8high), CD8SP (CD4ˉCD8+), and intermediate maturation subsets (CD4lowCD8low; CD4+CD8low; n = 5 mice per group). (B) Expression of CD8α and CD8β subunits within gated CD8α+ T cells in lymph nodes of OT1 TCR transgenic mice. The percentage of DP CD8α+CD8β+ T cells and the MFI for both markers is shown in the bar plots to the right (n = 3 mice per group). (C) CD69 and Annexin V expression was analyzed in Rras2+/+ and Rras2−/− DP OT1 TCR transgenic thymocytes after a 24-h incubation with the indicated concentrations of OVA peptide derivatives of different affinity. Annexin V values were adjusted using a semilog line function. Quantitative data are means ± SEM (n = 5). **, P < 0.005 (unpaired two-tailed Student’s t test). All experiments in this figure were repeated twice. n.s., not significant. reactivity of MOG tetramer with MOG-specific CD4+ T cells in compared with WT CD4 T cells (Fig. 7 D, right). Since TCR ex- immunized Rras2−/− mice than in WT controls. To confirm this pression assessed with an anti-CD3 antibody in MOG tetramer+ idea, we analyzed the MOG-responsive CD4+ T cell population CD4+ T cells was equal in WT and Rras2−/− mice (Fig. 7 D, center), within shorter times after immunization. In this case, we col- the smaller slope suggests that MOG-reactive CD4+ T cells lected the popliteal and inguinal draining lymph nodes and ex- in Rras2−/− mice have less affinity for their antigen than in amined the percentage of MOG tetramer+ CD4 T cells after a WT mice. brief MOG antigen-mediated expansion in vitro. The results showed a 3.5-fold reduction in the percentage of MOG tetramer+ Analysis of the TCR repertoire in Rras2−/− mice points to cells within the CD4+CD44+ population (Fig. 7 D, center) in TRAV4N-3 and TRAV4D-3 as Vα sequences biased toward Rras2−/− mice compared with WT. Of note, the adjustment to a autoimmunity line plot of MOG tetramer concentrations versus fluorescence A consequence of enhanced negative selection in Rras2−/− mice intensity reflected a smaller slope for staining of Rras2−/− might be manifested in a skewed TCR repertoire resulting from Martı́nez-Riaño et al. Journal of Experimental Medicine 2435 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 5. Deficient activation of the PI3K-Akt and Ras-ERK pathways in thymocytes from Rras2−/− mice. (A) Western blot of total thymocyte lysates stimulated with anti-CD3 antibody for the indicated times. Immunoblots were performed with the indicated phospho-specific antibodies. Immunoblots with antibodies to total ERK and/or CD3ζ were used as loading controls. A densitometric quantification of the intensity of the protein bands relative to the loading controls is shown to the right. A representative experiment out of four has been chosen for display. (B) Phosphoflow analysis of phospho-Akt (Ser473) and phospho-ERK (Thr202Tyr204) expression in thymocyte subpopulations after stimulation with anti-CD3 for the indicated time points. An overlay plot is shown in the middle panels illustrating changes in phospho-protein expression. Staining with an irrelevant rabbit antibody was used as negative control (C–; gray histograms). Quantitative data in the right panels are means ± SEM of triplicate values. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 (paired two-tailed Student’s t test). Significance was assessed for the entire dataset except for pERK in B, where it was assessed for time points 5–15 min. All experiments in this figure were repeated three times. n.s., not significant. Martı́nez-Riaño et al. Journal of Experimental Medicine 2436 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 6. Adoptive transfer of CD25ˉ CD4+ T cells from Rras2−/− mice to recipient Rag1−/− mice does not induce IBD symptoms. (A) Body weight loss of Rag1−/− mice transferred with Rras2+/+ (blue circles) or Rras2−/− (red squares) naive CD4 T cells or not transferred (gray circles). (B) Colon length, colon weight, and colon weight/length ratio of mice shown in A, sacrificed at day 46. (C) Total CD4+ T cell counts and expression of intracellular IFNγ, IL-17A, and Foxp3 by gated CD4+ T cells of spleens of adoptively transferred mice in A and B after 6 h of incubation with PMA plus ionomycin. Quantitative data in all panels are means ± SEM (n = 4–6 mice per group). *, P < 0.05; ***, P < 0.0005 (unpaired two-tailed Student’s t test). All experiments in this figure were repeated twice. n.s., not significant. a lower frequency of the most autoreactive T cell clones. To in WT T cells were detected at greatly reduced frequencies in study changes in the TCR repertoire, we purified total CD4+ Rras2−/− T cells (fivefold, eightfold, and fivefold, respectively). T cells from nonimmunized WT and Rras2−/− mice and se- Interestingly, the two VJ combinations have been associated quenced TRAV- and TRBV-encoding mRNAs after unbiased PCR with MOG reactivity in the EAE model (Kieback et al., 2016), and expansion. TRAV is the gene locus encoding Vα variable regions, therefore their diminished representation within the Rras2−/− and TRBV is the gene locus encoding Vβ variable regions. An T cell repertoire could explain the reduced susceptibility of average of 357,785 ± 47,449 (mean ± SD; n = 12) reads/mouse for Rras2−/− mice to MOG-induced EAE (Fig. 7 A). Furthermore, TRAV and of 365,643 ± 43,269 TRBV reads/mouse were obtained assuming they represent the high-affinity MOG clones, their from WT and Rras2−/− mice (Datasets 1 and 2). Sequence fre- scarce appearance could also explain the lower binding affinity quency comparison showed significant differences in the use of for MOG tetramer within the remaining MOG-specific reper- two Vβ regions and fourteen Vα regions, with a generally lower toire in these mice (Fig. 7 C). representation in Rras2−/− mice (Fig. 8 A). The analysis of the In addition to frequencies of VJ and VDJ recombinations, we usage of VJ combinations (TRA) and VDJ combinations (TRB) analyzed the frequencies of CDR3 loop amino acid sequences in showed prominent significant differences for certain combina- WT and Rras2−/− mice, which in TCRα reside at the VJ junction tions (Fig. 8 B). Two VJ (TRAV4D3-J22 and TRAV4N3-J22) and and in TCRβ at the VDJ junction (Glusman et al., 2001). The one VDJ combination (TRBV5-J1-4) that are relatively abundant CDR3α and CDR3β loops are especially relevant during thymic Martı́nez-Riaño et al. Journal of Experimental Medicine 2437 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 7. Rras2−/− mice are resistant to EAE in a MOG immunization-induced model. (A) Score and body weight evolution in Rras2+/+ (blue) and Rras2−/− (red) mice immunized with MOG. Scoring was as follows: 0, normal behavior; 1, weakness at the distal portion of the tail; 1.5, complete flaccidity of the tail; 2, moderate hind limb weakness; 2.5, severe hind limb weakness; 3, ataxia; 3.5, partial hind limb paralysis; 4, complete hind limb paralysis; 4.5, complete hind limb paralysis accompanied by muscle stiffness; 5, moribund state and hence sacrificed for humanitarian reasons. Quantitative data are means ± SEM (n = 5–6). **, P < 0.005. Score difference significance was assessed using a nonparametric Wilcoxon matched-pair signed rank test. Weight variations were compared using Martı́nez-Riaño et al. Journal of Experimental Medicine 2438 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 an unpaired two-tailed Student’s t test. This experiment was repeated three times. (B) Score and body weight evolution in lethally irradiated WT C57BL/6 mice reconstituted with bone marrow from either Rras2+/+ (blue) or Rras2−/− (red) mice and immunized with MOG. Quantitative data are means ± SEM (n = 8). *, P < 0.05; **, P < 0.005. Score difference significance was assessed using a nonparametric Wilcoxon matched-pair signed rank test; weight variations were compared using an unpaired two-tailed Student’s t test. This experiment was repeated twice. (C) Detection of inflammatory cytokine-producing T cells and T reg cells in spleen cells 7 d after immunization with MOG. For IFNγ and IL-17A production, spleen cells were stimulated overnight with PMA plus ionomycin and treated with brefeldin A before intracellular staining with anti-IFNγ and anti-IL17A antibodies. Quantitative data are means ± SEM (n = 3). In a different experiment, the presence of T reg cells was evaluated in spleen cells of MOG-immunized mice by staining with CD25 and intracellular staining with Foxp3. Quantitative data are means ± SEM (n = 9). *, P < 0.05; **, P < 0.005 (unpaired two-tailed Student’s t test). (D) Detection of MOG tetramer+ within the CD4+CD44+CD25+ cell population of splenocytes was evaluated 7 d after immunization and 48 h of in vitro stimulation with MOG peptide. The bar plots show the percentage of MOG tetramer+ cells within the CD4+ population and CD3 expression in CD4+ T cells. The scatter plot to the right shows the MFI of MOG tetramer binding within the positive population. A linear regression fit is shown as well as the slope of the line plots. Quantitative data are means ± SEM (n = 3). *, P < 0.05; ****, P < 0.00005 (unpaired two-tailed Student’s t test). Experiments in C and D were repeated three times. n.s., not significant. selection because they interact with self-peptides in the MHC-I suggesting a selection against T cells bearing a glycine residue in and MHC-II grooves (Garcia and Adams, 2005). This analysis position 4 of the CDR3α and a contribution of the 14–amino acid showed that various CDR3 sequences present in the WT reper- sequence with glycine in position 4 to self-peptide recognition. toire at a wide range of frequencies were strongly underrepre- Glycine in position 4 was found encoded by three different co- sented, or even absent, in the RRAS2-deficient T cell repertoire. dons (gga, ggg, ggt; Dataset 1), indicating that the sequence was These sequences might therefore belong to clones that were present in more than one TCR clone. Interestingly, the GASS more strongly negatively selected in the absence of RRAS2 CDR3α sequence was found in T cells of Rras2−/− mice, but not (Fig. 8 C). The TRAV sequence with the clearest overrepresen- in those of the WT, associated with another Vα sequence tation in WT mice versus Rras2−/− mice was a clone with the (TRAV4N4; Fig. 8 C). This might indicate that deletion of CDR3α sequence (CAAGASSGSWQLIF, from here on termed TCRα sequences in the absence of RRAS2 is dictated by both the “GASS”) encoded by the VJ combination TRAV4N3-TRAJ22, CDR3α sequence and the germline Vα-encoded CDR1α and which was present with the mean frequency of 0.5% in WT CDR2α sequences and that the latter CDRs also contribute to T cells and absent in Rras2−/− T cells (Fig. 8 C, arrow). Of note, autoreactivity. the CDR3 sequences most frequently found in Rras2−/− T cells To determine if the overabundance of T cell clones bearing a compared with their WT counterparts corresponded to clones of single CDR3α sequence in WT versus Rras2−/− CD4+ T cells was no or very low abundance in WT T cells (<0.001%; Fig. 8 C). This restricted to the TRAV4N-3 and TRAV4D-3 Vα regions or was suggests that those TCRs that are rare in WT mice could emerge more widespread, we studied the diversity of CDR3 sequences in the absence of RRAS2 because of enhanced positive selection. generated by each Vα. When comparing the number of different The comparison of preselection and post-selection TCR se- sequences generated by each Vα, we did not observe statistically quences has shown a shortening of CDR3β length as evidence of significant differences between WT and Rras2−/− cells (Fig. S4 D). TCR repertoire shaping during positive and negative selection A different issue is how homogenously a given sequence is (Matsutani et al., 2007). We found no significant differences in distributed within each Vα family. For this, we calculated the CDR3α and CDR3β length between the post-selection WT and Simpson index (Simpson, 1949), which measures the degree of RRAS2-deficient T cell repertoires, except for very short or very concentration when individuals are classified into types, ob- long ones, when all sequences were globally considered, re- taining a value of 1 when there is no diversity (all the sequences gardless of their V region usage (Fig. S4 A). However, we in the group are equal) and a value of 0 when the diversity is checked the TRAV4N-3 and TRAV4D-3 Vα regions in more de- maximal (all the sequences inside the group are different). We tail, given the strong reduction of these regions in the repertoire obtained an average Simpson index of 0.0441 (± 0.1124) for WT of Rras2-deficient animals. There was a strong significant effect sequences, and of 0.0326 (± 0.1069) for Rras2−/− sequences. This on the CDR3α length of these Vα regions (Fig. S4 B). Namely, the small difference was statistically significant (P = 0.016; paired frequency of T cell clones bearing the CDR3α sequence of 42 t test), suggesting that there is in general less diversity in the nucleotides (14 amino acids) of TRAV4N-3 and TRAV4D-3 Vα TCRα sequences, classified by their Vα, in WT than in Rras2−/− was reduced by two-thirds in Rras2−/− mice, whereas the fre- mice. In other words, in WT mice there is overrepresentation of quency of T cell clones bearing the 13-amino acid CDR3α asso- particular TCRα sequences that is not found, or found to a lower ciated to those Vα, was not significantly different from WT mice. extent, in Rras2−/− mice (Fig. 8 E, Fig. S4 E, and Dataset 3). This The analysis of the CDR3 sequence frequencies bearing any indicates that the effect of RRAS2 deficiency on the overrepre- of the two Vα regions showed that the sequence GASS, al- sentation of GASS-encoding clones (Fig. 8 D and Fig S4 C) is ready detected in Fig. 8 C, was present in 77% of the TRAV4N-3 extended to other clones expressing other CDR3α and other Vα T cell clones found in WT mice (Fig. 8 D), as well as in 70% of regions. the TRAV4D-3 T cell clones (Fig. S4 C). This sequence was vir- Finally, we aimed to determine if the differences detected by tually absent in T cells bearing any of the two Vα regions of RNA sequencing (RNaseq) in peripheral CD4 T cells of WT Rras2−/− mice. At the same time, a very similar sequence versus Rras2−/− mice regarding expression of TRAV4N-3 and (CAAEASSGSWQLIF) was found in T cells bearing TRAV4N-3 in TRAV4D-3 Vα sequences were already present in the thymus. To Rras2−/− mice at relatively high frequency (13%; Fig. 8 D), this end, we developed a qPCR strategy to detect the TRAV4N-3 Martı́nez-Riaño et al. Journal of Experimental Medicine 2439 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Martı́nez-Riaño et al. Journal of Experimental Medicine 2440 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Figure 8. Rras2−/−mice have a biased TCR repertoire in peripheral CD4+ T cells. (A) Bar graphs show significant differences in TCRα and TCRβ V region usage between WT (blue) and Rras2−/− (red) mice. Vα and Vβ families are ordered according to the P value (most significant to the left). Data are means ± SD (n = 6 mice per group). *, P < 0.05; **, P < 0.005 (unpaired two-tailed Student’s t test). (B) Analysis of TCR clone diversity based on TCRα VJ rearrangement (left panel) and TCRβ VDJ rearrangement (right panel). The graph shows the expression frequency of clones showing significant differences between WT and Rras2−/− mice (P < 0.05). Arrows emphasize two TCRα VJ rearrangements and one TCRβ VDJ rearrangement for which representation in the repertoire differs strongly between WT and Rras2−/− CD4+ T cells. (C) Clonotype diversity graphs of TCRα (left) and TCRβ (right) based on the expression frequency of CDR3 sequences. Purple and blue dots illustrate all CDR3 clones with an expression that is significantly different between both groups of animals (P < 0.05 and 0.01, respectively). To calculate log2 of the KO/WT frequency ratio (x axis), the 0 values for Rras2−/− and WT sequences were arbitrarily set as 0.00001 and 0.00003, respectively. (D) Frequency of specific CDR3α sequences for CD4 T cell clones bearing the TRAV4N-3 Vα region. (E) Representation of the Simpson Diversity Index calculated for all Vα families with a number of reads >100 in WT and Rras2−/− CD4+ T cells. A line has been hand drawn to indicate a diagonal of equal index values. Data in this figure result from the aggregation of two experiments (three mice of each genotype per group and per experiment) performed independently. n.s., not significant. and TRAV4D-3 sequences based on primers that partially anneal associated to TRAV4N-3 and TRAV4D-3 in Rras2−/− mice, but to the CDR1α and CDR3α regions (Fig. S5 A). With that set of positively selected when associated to TRAV4N-4-J22. There- primers, we found a significant difference of expression within fore, it is possible that the GASS sequence favors either negative the same sets of samples used for RNaseq (Fig. S5 B). Once we selection or positive selection depending on the context of the confirmed that the qPCR strategy could distinguish differences germline-encoded CDR1α and CDR2α sequences. The subsequent in expression of the TRAV4N-3 and TRAV4D-3 sequences, we corollary would be that positive and negative selections are the ran new samples of mRNA extracted from purified peripheral result of a global effect of the three CDRs on interaction with CD4+ T cells and from thymic CD4SP. We found that the self-pMHC in the thymus. The selection processes are hence not TRAV4N-3 and TRAV4D-3 sequences were already underrep- necessarily the result of a stepwise contribution first of germline resented in the thymus of Rras2−/− mice compared with the CDR1 and CDR2 interacting with positions in MHC α-helices for frequency in WT mice (Fig. S5 C). positive selection and then of the peptide-interacting CDR3 for negative selection (Huseby et al., 2005; Vrisekoop et al., 2014). The absence of particular VJ and VDJ combinations and CDR3 Discussion sequences in Rras2−/− mice, which on the other hand are highly We have found that RRAS2 plays an important role in shaping abundant in WT mice, reinforces the idea that RRAS2 helps to the T cell repertoire in the thymus. In the absence of RRAS2, DP set the threshold for negative selection in the thymus. RRAS2 thymocytes undergo a more stringent negative selection, re- could do so by providing a pro-survival signal for those DP sulting in peripheral resistance to autoimmunity manifested in thymocytes that bear a TCR affinity just above this threshold. two models of disease. While autoantigens in the adoptive This idea is sustained by the enhanced deletion of AND TCR- transfer IBD model are unknown, in the EAE model the disease is bearing thymocytes in the presence of the H-2k haplotype, by unleashed by breaking self-tolerance through immunization the overexpression of Nur77 and other markers of negative se- with a peptide derived from MOG protein in the presence lection, and by the lack of inhibition of transcription of genes of strong adjuvants. Interestingly, we found that a faster associated with positive selection. In this regard, the analysis of improvement from neurological impairment symptoms in sequence diversity does not show impoverishment or enrich- Rras2−/−mice was correlated with a lower affinity of MOG- ment in sequence diversity for TCRα in Rras2−/− versus WT CD4+ reactive CD4+ T cells for MOG tetramers in mice. Further- T cells, suggesting that the overall effect of RRAS2 deficiency is more, the comparative analysis of TRAV and TRBV repertoires not that of reducing or increasing positive selection. By contrast, in peripheral T cells from Rras2−/− and WT mice has allowed us the Simpson analysis of diversity shows that there is overrep- to identify two Vα regions (TRAV4D-3 and TRAV4N-3), one Jα resentation of certain CD4 T cell clones in WT mice and not in region (J22), and the 14–amino acid CDR3α GASS sequence as Rras2−/− mice, of which those that express the GASS sequence in TCRα combinations that are strongly disfavored in the absence the TRAV4N-3 and TRAV4D-3 Vα families are just examples. of RRAS2. TRAV4N-3 and TRAV4D-3 sequences have been pre- In our opinion, the overrepresentation of independent clones viously linked to MOG recognition (Kieback et al., 2016), al- bearing certain TCRα sequences in WT mice is due to the en- though they are not always linked to J22 or to the GASS hanced signaling fitness of thymocytes bearing those sequences. sequence. Interestingly, one of the MOG-reactive T cell clones During immune responses in mice (Malherbe et al., 2004) and bears a TRAV4ND-3 Vα sequence associated to J22 with a CDR3α humans (Price et al., 2005), the T cell repertoire becomes re- sequence (CAADSSGSWQLIF, sequence N5-575; Fillatreau et al., stricted to a few dominant clones that have higher affinity for 2012) similar to GASS, suggesting that the latter might be in- their ligand. Skewing of the preselection repertoire, based on volved in MOG recognition, as well. Another interesting finding, quantitative parameters of the TCR self-pMHC interactions, also in our opinion, is that the GASS sequence, highly abundant in occurs in the thymus (Savage and Davis, 2001). This indicates WT mice but absent in Rras2−/− mice when associated to that the strength of the TCR–MHC interaction is a driving force TRAV4N-3-J22, is present in Rras2−/− but not in WT mice when in shaping the immature and mature T cell repertoire. Our ob- associated to TRAV4N-4-J22. These data suggest that T cells servation that abundant clones in WT mice, which include bearing the GASS sequence are negatively selected when this is previously identified autoreactive clones, disappear in the Martı́nez-Riaño et al. Journal of Experimental Medicine 2441 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 thymus of Rras2−/− mice and the finding that these latter mice PI3K-Akt pathway in negative selection could be driven by Akt- are more resistant to experimental autoimmune diseases sup- mediated phosphorylation and degradation of the pro-apoptotic port the hypothesis that strongly represented clones in the transcription factor involved in negative selection, Nur77 thymus of nonmanipulated WT mice are enriched for auto- (Masuyama et al., 2001; Huo et al., 2010). Therefore, RRAS2 reactive T cells. As such, those clones are deleted in the absence deficiency would lead to low PI3K-Akt activity and enhanced of RRAS2 due to increased negative selection. susceptibility to apoptosis induced by relatively high-affinity The reduced autoimmune capacity of the Rras2−/− T cell TCR interactions with self-pMHC complexes. The pronounced repertoire could in principle also be due to an impairment of defect in Akt phosphorylation detected in Rras2−/− thymocytes positive selection of low-affinity autoreactive clones. The lower demonstrates that RRAS2 is required for the activation of the TCR-mediated signaling capacity of the Rras2−/− T cell pre- PI3K-Akt pathway by TCR triggering in thymocytes. However, cursors might prohibit them, as well as self-tolerant low-affinity although less evident, we have also found that Rras2−/− thymo- clones, from reaching the minimally required positive selec- cytes are deficient in maintaining a sustained ERK phosphoryl- tion signaling threshold. We consider this scenario less likely ation in response to TCR triggering. Interestingly, positive for three reasons. First, the MOG-specific T cell repertoire of selection was associated with weak but prolonged ERK phos- Rras2−/− mice has a lower affinity for this antigen compared with phorylation, whereas negative selection was associated with a the T cell repertoire in WT mice, showing that signaling in ab- rapid, transitory peak of ERK phosphorylation (reviewed in sence of RRAS2 permits inclusion of such low-affinity clones in Teixeiro and Daniels, 2010). Therefore, it could be that enhanced the mature T cell repertoire. Second, the two TCR-transgenic negative selection in the absence of RRAS2 is due to defective models of positive selection we tested (OT-1; AND on a pure sustained ERK phosphorylation rather than to defective activa- H-2b background) show no obvious defects in this process in tion of the PI3K-Akt pathway. Nonetheless, it is also known that Rras2−/− mice. Finally, the enhanced functional response of PI3K activates ERK and that both Ras-ERK and PI3K-Akt path- Rras2−/− OT-1 thymocytes to positively selecting ligands com- ways are intensely interconnected (Mendoza et al., 2011). At this pared with WT thymocytes indicates that it is not the ability to stage, it is not possible to ascribe the protective effect of RRAS2 respond to weak ligands that is inhibited but that it may actually during negative selection to either the Raf-ERK or the PI3K-Akt change the fate of the thymocytes from positive selection to pathway, although we favor a model in which it is the integra- negative selection. tion of the global effect on both pathways that determines the The nature of the signaling pathways activated by RRAS2 final outcome. Of note, if such balance determines positive and involved in protection against negative selection is not fully negative selection, the closely related small GTPases HRAS and understood, although there are some clues derived from previ- NRAS do not seem to significantly contribute to this equilib- ous knowledge. RRAS2 is an activator of the PI3K-Akt pathway rium, since simultaneous germline deletion of both genes did not rather than the Raf-ERK pathway, and it has been previously cause detectable effects on positive and negative selections described to deliver pro-survival signals to mature T and B cells (Iborra et al., 2011). Accordingly, we have identified a new type (Delgado et al., 2009). The role of the PI3K-Akt pathway during of regulator of selection thresholds in the thymus, different in thymic differentiation of T cells has been investigated mainly on class from other proteins identified as key players, such as the pre-TCR–driven transition from DN to DP thymocytes, Themis, a possible adaptor for tyrosine phosphatases (Fu et al., where the PI3K-Akt pathway is required to maintain thymocyte 2013; Paster et al., 2015); Bim, a pro-apoptotic protein acting survival during β-selection rather than thymocyte proliferation through the mitochondrial pathway; and Nur77, a transcrip- (Juntilla and Koretzky, 2008). In contrast, we have not detected tional regulator of negative selection. Since RRAS2 directly binds a significant effect on the DN populations in Rras2−/− mice, the TCR (Delgado et al., 2009), one could speculate that it is suggesting that RRAS2 does not contribute in an important upstream of the other players. Further work will help to de- manner to pre-TCR signaling. The role of the PI3K-Akt pathway termine the existence of such functional connections. beyond the DP stage has not been studied in detail. An early work described that deletion of Pten, a negative regulator of PI3K activity, leads to increased thymic cellularity due to a Materials and methods possible negative selection defect (Suzuki et al., 2001). Inter- Mice estingly, the overexpression of Pten beyond the β-selection Rras2−/− mice were generated as previously described (Delgado checkpoint resulted in reduced DP and SP thymic populations, et al., 2009). These mice were crossed with AND TCRTg mice which was interpreted as a defective maturation to DP of a expressing I-Ab or I-Ek MHC class II (Kaye et al., 1989) in order to transitional intermediate SP population (Xue et al., 2008). generate Rras2−/− ANDTg I-Ab/I-Ek. Rras2−/− mice were also However, the idea that reduced DP and SP thymocytes was due crossed with mice transgenic for the OT2 TCR specific for a to enhanced negative selection was not discarded. Likewise, a peptide corresponding to residues 323–339 of chicken OVA double deletion of the p110δ and p110γ catalytic subunits of presented by I-Ab (Barnden et al., 1998). C57BL/6 mice bearing PI3Kδ and PI3Kγ, respectively, resulted in massive death at the the pan-leukocyte marker allele CD45.1 were kindly provided by DP stage, although pre-TCR function and DP generation re- Dr. Carlos Ardavı́n (Centro Nacional de Biotecnologı́a, Madrid, mained unperturbed (Swat et al., 2006). These data point to a Spain). Cd3e−/− mice, deficient in the expression of CD3ε were role for the PI3K pathway in the survival of thymocytes beyond obtained from Jackson Laboratories (DeJarnette et al., 1998). the DP stage. Mechanistically, the anti-apoptotic role of the Mice homozygous for the Rag1tm1Mom mutation lack both T and Martı́nez-Riaño et al. Journal of Experimental Medicine 2442 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 B cells (Mombaerts et al., 1992) and were kindly provided by Dr. marrow cells from Rras2+/+ or Rras2−/− ANDTg TCR mice ex- César Cobaleda (Centro de Biologı́a Molecular Severo Ochoa, pressing only I-Ab were harvested and injected in the irradiated Madrid, Spain). All animals were backcrossed to the C57BL/6J receptor mice. Thymuses of reconstituted animals were ana- background (Envigo) for ≥10 generations. For all in vivo ex- lyzed by flow cytometry 6–8 wk after reconstitution. periments, age (5–8 wk) and sex were matched between the For mixed bone marrow competition experiments, bone Rras2+/+ (WT) and Rras2−/− (KO) mice. Mice were maintained marrow cells from Rras2+/+ (CD45.2+) or Rras2−/− (CD45.2+) were under specific pathogen-free conditions in the animal facility of mixed with bone marrow cells in a 50:50 ratio of C57BL/6 the Centro de Biologı́a Molecular Severo Ochoa in accordance CD45.1+ mice. Mixed bone marrow cells were used to reconsti- with applicable national and European guidelines. All animal tute lethally irradiated (10 Gy) C57BL/6 CD45.1+ receptor mice. procedures were approved by the ethical committee of the Thymuses of reconstituted mice were collected and analyzed by Centro de Biologı́a Molecular Severo Ochoa. flow cytometry 8 wk after reconstitution. Antibodies and reagents Analysis of T cell activation The following antibodies were used: anti-mouse CD45R-V450 Thymocytes were resuspended in RPMI plus 20 mM Hepes and -biotin -APC (RA3-6B2), CD4-PerCP -647 -FITC (RM4-5), CD8- left in starving conditions for 1 h. Cells were stimulated at dif- biotin -V405 -PerCP -647 (53–6.7), CD11b-biotin (M1/70), puri- ferent time points with soluble anti-CD3 (2C11) antibody (0, 1, 5, fied CD16/32 (2,4G2), CD19–PE-Cy7 (1D3), CD25-APC (3C7), 15, and 30 min) at 37°C. CD43-biotin (S7), CD45.1–APC-Cy7 (A20), CD45.2-APC (104), When T cell stimulation was analyzed by immunoblotting, Gr1-biotin (RB6-8C5), CD44-PE (IM7), TCRβ-FITC (H57-597), after stimulation, cells were lysed in Brij96 lysis buffer con- CD3ε-PerCP (2C11), Vβ3-biotin -PE (KJ25), CD5-PE -biotin taining protease and phosphatase inhibitors (1% Brij96, 140 mM (53–7.3), IL-17a–PerCP (TC11-18H10), CD69-FITC -PerCP (H1.2F3), NaCl, 10 mM Tris–HCl [pH 7.8], 10 mM iodoacetamide, 1 mM and NK1.1-biotin from BD Pharmigen; anti-mouse F4/80–biotin PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM sodium or- (BM8), H2-Kk–V405 (AF3-12.1.3), Foxp3-PE (NRRF-30), and thovanadate, 20 mM sodium fluoride, and 5 mM MgCl2). After Nur77-PE (12.14) from eBioscience; anti-mouse IFNγ–APC from removing the nuclei by low-speed centrifugation, cell lysates Milteny; anti-mouse Vα2–PerCP (B20.1) from BioLegend; rabbit were resolved by SDS-PAGE and immunoblotted using standard anti-mouse pERK (T202 Y204), pAKT (S473), pS6 (S240), total protocols. The membranes were probed with anti-phospho AKT, ERK, and total CD3ε from Cell Signaling. anti-phospho ERK, anti-phospho p70S6, or anti-total ERK anti- I-Ab MOG3555 Tetramer-PE was acquired from MBL. I-Ab bodies (Cell Signaling), visualizing the protein bands by ECL OVA329-337 Tetramer-APC was a generous donation of the Na- (Pierce). tional Institutes of Health Tetramer Core Facility. OVAp (SIINFEKL), When T cell stimulation was analyzed by flow cytometry, Q4R7, Q4H7 (SIIQFEHL), E1 (EIINFEKL), and classII-OVA after stimulation, cells were fixed in 4% paraformaldehyde. (AAHAEINEA) peptides were from Anaspec. CellTrace Violet First, cells were stained extracellularly with anti-CD4 and anti- (CTV; C34557) was from Invitrogen. OVA (A5503) and LPS CD8 antibodies in order to differentiate the four different thy- (L2630) were from Sigma-Aldrich. mocyte subpopulations, and afterwards permeabilized with 0.1% NP-40 and incubated with rabbit anti-phosho ERK and anti- Cell preparation phosho AKT (Cell Signaling) followed by an anti-rabbit Ig The thymus, lymph nodes, and spleen from 5–8-wk-old mice Alexa 488 antibody (Jackson ImmunoReasearch). Phosphoryla- were homogenized with 40 µm strainers and washed in PBS tion levels were quantified by flow cytometry. containing 2% (vol/vol) FBS. Spleen cells were resuspended for 3 min in AcK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM In vitro apoptosis assay EDTA, pH 7.2–7.4) to lyse the erythrocytes and then washed in Splenocytes from Cd3e−/− mice were incubated for 2 h with PBS 2% FBS. different concentrations of OVAp, Q4R7, Q4H7, and E1 peptides For in vitro cultures, cells were maintained in RPMI with 10% based on Ref (Daniels et al., 2006). Afterwards, cells were FBS supplemented with 2 mM L-glutamine, 100 U/ml penicillin, washed, resuspended in RPMI medium, and plated in a 96-well 100 U/ml streptomycin, 20 µM β-mercaptoethanol, and 10 mM plate together with 0.2 × 106 thymocytes from Rras2+/+ or sodium pyruvate. Rras2−/− OTI mice. 20 h later, cells were stained for CD4, CD8, Vα2, and CD69. Annexin V labeling on the membrane of thy- Flow cytometry mocytes was measured by staining with an anti-Annexin V (BD) Mouse single-cell suspensions were incubated with fluorescence- following the manufacter’s instructions. labeled antibodies for 30 min at 4°C after blocking Fc receptors using anti-CD16/32 antibody. Afterwards, cells were washed in In vivo T cell proliferation PBS+1%BSA, and data were collected on a FACS Canto II. Analyses CD4 T cells from lymph nodes and spleens of Rras2+/+ and were performed using the FlowJo program. Rras2−/− OT2tg mice, expressing the CD45.2 allele, were purified by negative selection using a mix of biotinylated antibodies: Bone marrow reconstitution anti-B220, anti-CD8, anti-NK1.1, anti-CD11b, anti-Gr1, and anti- ANDTg TCR expressing I-Ek MHC class II mice were irradiated F4/80. Afterwards, stained cells were incubated with strepta- lethally with 1,000 rads the day before reconstitution. Bone vidin beads (Dynabeads Invitrogen) for 30 min and separated Martı́nez-Riaño et al. Journal of Experimental Medicine 2443 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 using a Dynal Invitrogen Beads Separator. Proliferation of OT2 muscle stiffness; 5, moribund state and hence sacrificed for cells was assessed using CTV labeling as specified by the man- humanitarian reasons. ufacturer (Thermo Fisher Scientific). A total of 106 CTV-labeled Alternatively, mice were sacrificed 7 d after immunization, OT2 T cells were inoculated intravenously into CD45.1+ receptor before clinical disease signals appeared, in order to analyze the mice, and 24 h later they were immunized intraperitoneally presence of MOG+ activated T cells in the popliteal lymph nodes with 200 µg OVA plus 50 µg LPS in saline buffer. 5 d after by staining the cells with I-Ab MOG35-55 tetramer (National In- immunization, splenocytes were analyzed by flow cytometry for stitutes of Health tetramer core facility) at different dilutions for CTV dilution within the transferred OT2 T cells. 30 min. Mice were also analyzed 30 d after immunization, at which time activated MOG+ T cells were also assessed in the In vitro T cell activation spleen by flow cytometry. A total of 0.5 × 106 splenocytes from Rras2+/+ and Rras2−/− mice To test the T cell–intrinsic dependency of EAE resistance, were plated in 96-well plates that had been preincubated with 8-wk-old female C57BL/6 mice were lethally irradiated (10 Gy) anti-CD3 antibody (20 µg/ml) with or without 1 µg/ml soluble and reconstituted with 6 × 106 of a bone marrow mixture of cells anti-CD28. 24 h later, CD69 expression was analyzed by flow containing 80% bone marrow cells from female Cd3e−/− mice and cytometry. 20% bone marrow cells from either female Rras2+/+ or Rras2−/− mice. At 8 wk after reconstitution, mice were immunized and RT-PCR scored as above. DP thymocytes were sorted (using a FACSAria Fusion BSC II) from Rras2+/+ and Rras2−/− mice, and their RNA was isolated Immunization with class II-restricted OVA peptide using the RNAeasy Plus Mini Kit (QIAGEN). cDNA was syn- 8-wk-old female Rras2+/+ and Rras2−/− mice were immunized by thesized with SuperScript III (Invitrogen) using Oligo-dT pri- inoculation of 25 µl of a PBS solution containing 100 µg OVA mers. RT-qPCR was performed in triplicate using the reverse 329–337 peptide (AAHAEINEA) and 15 µg LPS (Sigma-Aldrich) transcription reaction with SYBR Green PCR Master Mix and into each hind limb footpad. Mice were euthanized 7 d after gene-specific primers in an ABI 7300 Real Time PCR System. immunization. Obtained cycle threshold (Ct) values were used to calculate mRNA levels relative to hypoxanthine-guanine phosphoribo- Analysis of TCR repertoire syltransferase and GAPDH expression using the 2-ΔCt method. CD4+ T cells from the spleens of six Rras2+/+ and six Rras2−/− 8- wk-old female and male mice (in equal proportion in both IBD groups) were purified by negative selection using a mix of bi- A total of 0.5 × 106 sorted CD4+ CD25− naive T cells from spleens otinylated antibodies: anti-B220, anti-CD8, anti-NK1.1, anti- of Rras2+/+ and Rras2−/− mice were inoculated intraperitoneally CD11b, anti-Gr1, and anti-F4/80. Afterwards, stained cells were into age-matched Rag1tm1Mom mice. Total body weight was incubated with streptavidin beads (Dynabeads Invitrogen) for monitored one to two times per week, and mice were sacrificed 7 30 min and separated using the Dynal Invitrogen Beads Sepa- wk after initiation of the experiment. The colon was removed rator. mRNA was extracted from purified CD4+ T cells using the and flushed with PBS, and the length and weight was measured RNAeasy Plus Mini Kit (QIAGEN). cDNA libraries were prepared from the rectum to the cecum. Spleens were removed, and cells using the 59RACE-based protocol (i.e., onf read covers CDR3 and were counted and analyzed by flow cytometry to detect the its surroundings, and another one covers the 59UTR and percentage of IL-17– and IFNγ-producing CD4+ T cells after downstream sequence of the V gene) with the SMARTer Mouse treatment with PMA (50 ng/ml) plus ionomycin (750 µg/ml) for TCR α/β Profiling Kit (Takara) following the manufacturer’s 12 h in the presence of 10 µg/ml brefeldin A for the final 4 h. instructions. Briefly, the reverse transcription reaction adds nontemplate nucleotides to the first strand of the cDNA that are EAE used as template nucleotides for posterior PCR oligonucelotides. EAE was induced in female Rras2+/+ and Rras2−/− C57BL/6 mice Afterwards, a semi-nested PCR with the first PCR amplification (6–8 wk old; 20 g body weight) by subcutaneously injecting a of the full-length variable region of the TCR is performed, fol- total of 150 µg MOG35–55 (Espikem) emulsified in Freund’s lowed by a subsequent round of PCR to further amplify full- complete adjuvant (Sigma-Aldrich) supplemented with Myco- length sequences of V(D)J variable regions of TCRα and/or bacterium tuberculosis (1 mg/ml; H37Ra strain from Difco) into TCRβ subunits and to incorporate adapter sequences for Illu- both femoral regions. 200 ng pertussis toxin (Sigma-Aldrich) mina sequencing. per mouse were immediately injected intraperitoneally. A sec- The cDNA libraries were sequenced on an Illumina MiSeq ond dose of pertussis toxin was administered 48 h after immu- (2 × 300 nt). The quality of sequencing reads was assessed using nization. The animals were weighted and inspected for clinical FastQC. To extract CDR3 sequences; to determine V, D, and J signs of disease. Disease severity of EAE was assessed according genes; and to assemble clonotypes by CDR3 sequences, the raw to the EAE severity scale: 0, normal behavior; 1, weakness at the sequencing reads were processed using the MiXCR v2.1.9 soft- distal portion of the tail; 1.5, complete flaccidity of the tail; 2, ware (Bolotin et al., 2015) programs Align (with the parameters moderate hind limb weakness; 2.5, severe hind limb weakness; -OvParameters.geneFeatureToAlign = VTranscript --species 3, ataxia; 3.5, partial hind limb paralysis; 4, complete hind limb mmu --chain TRA,TRB), Assemble (with the parameters paralysis; 4.5, complete hind limb paralysis accompanied by -OseparateByJ = true -OseparateByV = true), and exportClones (with Martı́nez-Riaño et al. Journal of Experimental Medicine 2444 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 the parameters --filter-out-of-frames --filter-stops). The clones Institutes of Health (Bethesda, MA) tetramer core facility for present (containing at least one read) in less than three samples provision of MHC–peptide complexes, the Centre for Genomic of either six Rras2+/+ or six Rras2−/− mice were filtered out. Regulation Genomics Unit for sequencing T-cell repertoire data, Further analysis of clones was conducted using in-house written and the Centre or Genomic Regulation Bioinformatics Unit for R-scripts and VDJtools v1.1.7 (Shugay et al., 2015). providing support in sequencing data analysis. This work was funded by grants from Comisión Interminis- Determination of TRAV4N3-J22 and TRAV4D3-J22 expression terial de Ciencia y Tecnologı́a (SAF2016-76394-R to B. Alarcon), by qPCR analysis the “Comunidad de Madrid” (S2017/BMD-3671 to M. Fresno and Purified CD4+ T cells and CD44SP from lymph nodes and thy- B. Alarcon), and the European Research Council (ERC 2013- mus, respectively, were obtained by cell sorting. RNA and cDNA Advanced Grant 334763 “NOVARIPP” to B. Alarcon). The Centre extraction from these cells was performed according to the for Genomic Regulation acknowledges support of the Spanish manufacturers’ instructions (Qiagen kit #74136 and Invitrogen Ministry of Economy and Competitiveness, “Centro de Ex- kit #11752-050, respectively). qPCR was performed at 63.6°C celencia Severo Ochoa,” the Centres de Recerca de Catalunya using specific primers for TRAV4N3 and TRAVD3 sequences Program/Generalitat de Catalunya, and the Centro de Biologı́a (Fw: 59-AGATGCAATTTTTCTATCGCTGC-39; Rv: 59-TCCAAAGA Molecular Severo Ochoa to the Fundación Ramón Areces. TGAGTTGCCAGCT-39) and GAPDH. Relative gene expression The authors declare no competing financial interests. levels of the sequences were obtained by normalizing the ob- Author contributions: A. Martı́nez-Riaño designed and per- tained Ct to GAPDH. formed the research, analyzed the data, and wrote the manuscript. E.R. Bovolenta and V.L. Boccasavia assisted with analysis of TCR Diversity analysis sequence expression in the thymus. C.L. Oeste edited the manu- Sequence diversity among the clones generated by each V type script. J. Ponomarenko analyzed TCR repertoire sequencing data, gene were measured using the Simpson index (Simpson, 1949). prepared related figures, and edited the manuscript. D. Abia Sequences from Rras2+/+ and Rras2−/− samples were classified contributed to the figures presenting the sequencing data analysis. independently according to their V type, and the Simpson index M. Fresno and H.M. van Santen helped to write the manuscript was calculated, with an in-house perl script, for each group, and supervised research. B. Alarcon supervised and designed the whenever there were >100 sequences for the WT and the research, analyzed the data, and wrote the manuscript. Rras2−/− for the group. Submitted: 17 October 2018 Statistical analysis Revised: 29 March 2019 Statistical parameters including the exact value of n and the Accepted: 19 June 2019 means ± SD or SEM are described in the figures and figure legends. The nonparametric Wilcoxon–Mann–Whitney U test and the parametric Student’s t test were used as indicated to References assess the difference in means. Alcover, A., B. Alarcon, and V. Di Bartolo. 2018. Cell biology of T cell receptor expression and regulation. Annu. Rev. Immunol. 36:103–125. Data availability Azzam, H.S., A. Grinberg, K. Lui, H. Shen, E.W. Shores, and P.E. Love. 1998. CD5 expression is developmentally regulated by T cell receptor (TCR) Raw sequencing data are available in the National Center for signals and TCR avidity. J. Exp. Med. 188:2301–2311. https://doi.org/10 Biotechnology Information Sequence Read Archive under the .1084/jem.188.12.2301 accession number PRJNA488012. Barbee, S.D., and J. Alberola-Ila. 2005. Phosphatidylinositol 3-kinase regu- lates thymic exit. J. Immunol. 174:1230–1238. https://doi.org/10.4049/ jimmunol.174.3.1230 Online supplemental material Barnden, M.J., J. Allison, W.R. Heath, and F.R. Carbone. 1998. Defective TCR Fig. S1 shows the expression of TCR and coreceptors as well as expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory el- the presence of nT reg cells in WT and Rras2−/− mice. Fig. S2 ements. Immunol. Cell Biol. 76:34–40. https://doi.org/10.1046/j.1440-1711 shows the response of WT and Rras2−/− T cells to TCR-triggering .1998.00709.x stimuli. Fig. S3 shows the distribution of CDR3 lengths in the Bendelac, A., P. Matzinger, R.A. Seder, W.E. Paul, and R.H. Schwartz. 1992. Activation events during thymic selection. J. Exp. Med. 175:731–742. TCRα and TCRα chains. Fig. S4 shows the representation of T cell https://doi.org/10.1084/jem.175.3.731 clones bearing particular TCRα sequences in WT and Rras2−/− Bolotin, D.A., S. Poslavsky, I. Mitrophanov, M. Shugay, I.Z. Mamedov, E.V. mice. Fig S5 shows the sequences of annealing primers and qPCR Putintseva, and D.M. Chudakov. 2015. MiXCR: software for compre- hensive adaptive immunity profiling. Nat. Methods. 12:380–381. https:// data showing expression of particular TCRα sequences. Dataset doi.org/10.1038/nmeth.3364 1 shows TRA sequences per mouse. Dataset 2 shows TRB se- Borroto, A., I. Arellano, E.P. Dopfer, M. Prouza, M. Suchànek, M. Fuentes, A. quences per mouse. Dataset 3 shows an analysis of TRA sequence Orfao, W.W. Schamel, and B. Alarcón. 2013. Nck recruitment to the TCR diversity within Vα families. required for ZAP70 activation during thymic development. J. Immunol. 190:1103–1112. https://doi.org/10.4049/jimmunol.1202055 Brändle, D., S. Müller, C. Müller, H. Hengartner, and H. Pircher. 1994. Reg- ulation of RAG-1 and CD69 expression in the thymus during positive Acknowledgments and negative selection. Eur. J. Immunol. 24:145–151. https://doi.org/10 .1002/eji.1830240122 We are indebted to Cristina Prieto, Valentina Blanco, and Tania Cantrell, D. 2015. Signaling in lymphocyte activation. Cold Spring Harb. Per- Gómez for their expert technical assistance, the National spect. Biol. 7:a018788. https://doi.org/10.1101/cshperspect.a018788 Martı́nez-Riaño et al. Journal of Experimental Medicine 2445 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Choi, S., R. Cornall, R. Lesourne, and P.E. Love. 2017. THEMIS: two models, Derived Regulatory T Cells Are Positively Selected on Natural Self- different thresholds. Trends Immunol. 38:622–632. https://doi.org/10 Antigen through Cognate Interactions of High Functional Avidity. Im- .1016/j.it.2017.06.006 munity. 44:1114–1126. https://doi.org/10.1016/j.immuni.2016.04.018 Daniels, M.A., E. Teixeiro, J. Gill, B. Hausmann, D. Roubaty, K. Holmberg, G. Klein, L., B. Kyewski, P.M. Allen, and K.A. Hogquist. 2014. Positive and Werlen, G.A. Holländer, N.R. Gascoigne, and E. Palmer. 2006. Thymic negative selection of the T cell repertoire: what thymocytes see (and selection threshold defined by compartmentalization of Ras/MAPK don’t see). Nat. Rev. Immunol. 14:377–391. https://doi.org/10.1038/ signalling. Nature. 444:724–729. https://doi.org/10.1038/nature05269 nri3667 DeJarnette, J.B., C.L. Sommers, K. Huang, K.J. Woodside, R. Emmons, K. Katz, Kortum, R.L., A.K. Rouquette-Jazdanian, and L.E. Samelson. 2013. Ras and E.W. Shores, and P.E. Love. 1998. Specific requirement for CD3epsilon extracellular signal-regulated kinase signaling in thymocytes and in T cell development. Proc. Natl. Acad. Sci. USA. 95:14909–14914. T cells. Trends Immunol. 34:259–268. https://doi.org/10.1016/j.it.2013.02 https://doi.org/10.1073/pnas.95.25.14909 .004 Delgado, P., B. Cubelos, E. Calleja, N. Martı́nez-Martı́n, A. Ciprés, I. Mérida, C. Malherbe, L., C. Hausl, L. Teyton, and M.G. McHeyzer-Williams. 2004. Clonal Bellas, X.R. Bustelo, and B. Alarcón. 2009. Essential function for the selection of helper T cells is determined by an affinity threshold with no GTPase TC21 in homeostatic antigen receptor signaling. Nat. Immunol. further skewing of TCR binding properties. Immunity. 21:669–679. 10:880–888. https://doi.org/10.1038/ni.1749 https://doi.org/10.1016/j.immuni.2004.09.008 Dower, N.A., S.L. Stang, D.A. Bottorff, J.O. Ebinu, P. Dickie, H.L. Ostergaard, Martı́nez-Martı́n, N., R.M. Risueño, A. Morreale, I. Zaldı́var, E. Fernández- and J.C. Stone. 2000. RasGRP is essential for mouse thymocyte differ- Arenas, F. Herranz, A.R. Ortiz, and B. Alarcón. 2009. Cooperativity entiation and TCR signaling. Nat. Immunol. 1:317–321. https://doi.org/10 between T cell receptor complexes revealed by conformational mutants .1038/79766 of CD3epsilon. Sci. Signal. 2:ra43. https://doi.org/10.1126/scisignal Falk, I., G. Nerz, I. Haidl, A. Krotkova, and K. Eichmann. 2001. Immature .2000402 thymocytes that fail to express TCRbeta and/or TCRgamma delta pro- Masuyama, N., K. Oishi, Y. Mori, T. Ueno, Y. Takahama, and Y. Gotoh. 2001. teins die by apoptotic cell death in the CD44(-)CD25(-) (DN4) subset. Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis. Eur. J. Immunol. 31:3308–3317. https://doi.org/10.1002/1521-4141(200111) J. Biol. Chem. 276:32799–32805. https://doi.org/10.1074/jbc.M105431200 31:11<3308::AID-IMMU3308>3.0.CO;2-5 Matsutani, T., T. Ohmori, M. Ogata, H. Soga, S. Kasahara, T. Yoshioka, R. Fayard, E., G. Moncayo, B.A. Hemmings, and G.A. Holländer. 2010. Phos- Suzuki, and T. Itoh. 2007. Comparison of CDR3 length among thymo- phatidylinositol 3-kinase signaling in thymocytes: the need for strin- cyte subpopulations: impacts of MHC and BV segment on the CDR3 gent control. Sci. Signal. 3:re5. https://doi.org/10.1126/scisignal.3135re5 shortening. Mol. Immunol. 44:2378–2387. https://doi.org/10.1016/j Fillatreau, S., W. Uckert, E. Kieback, S. Anderton, V. Lampropoulou, U. .molimm.2006.10.026 Stervbo, and M. Bunse. 2012. TCR transgenic mouse model for immune Mendoza, M.C., E.E. Er, and J. Blenis. 2011. The Ras-ERK and PI3K-mTOR disease. European Patent Application 12175113.5, priority date July 5, pathways: cross-talk and compensation. Trends Biochem. Sci. 36: 2012. 320–328. https://doi.org/10.1016/j.tibs.2011.03.006 Fischer, A.M., C.D. Katayama, G. Pagès, J. Pouysségur, and S.M. Hedrick. Mendoza, P., N. Martı́nez-Martı́n, E.R. Bovolenta, D. Reyes-Garau, P. Her- 2005. The role of erk1 and erk2 in multiple stages of T cell development. nansanz-Agustı́n, P. Delgado, M.D. Diaz-Muñoz, C.L. Oeste, I. Fernán- Immunity. 23:431–443. https://doi.org/10.1016/j.immuni.2005.08.013 dez-Pisonero, E. Castellano, et al. 2018. R-Ras2 is required for germinal Fu, G., J. Casas, S. Rigaud, V. Rybakin, F. Lambolez, J. Brzostek, J.A. Hoerter, center formation to aid B cells during energetically demanding pro- W. Paster, O. Acuto, H. Cheroutre, et al. 2013. Themis sets the signal cesses. Sci. Signal. 11:11. https://doi.org/10.1126/scisignal.aal1506 threshold for positive and negative selection in T-cell development. Merkenschlager, M., D. Graf, M. Lovatt, U. Bommhardt, R. Zamoyska, and Nature. 504:441–445. https://doi.org/10.1038/nature12718 A.G. Fisher. 1997. How many thymocytes audition for selection? J. Exp. Garcia, K.C., and E.J. Adams. 2005. How the T cell receptor sees antigen--a Med. 186:1149–1158. https://doi.org/10.1084/jem.186.7.1149 structural view. Cell. 122:333–336. https://doi.org/10.1016/j.cell.2005.07 Mingueneau, M., T. Kreslavsky, D. Gray, T. Heng, R. Cruse, J. Ericson, S. .015 Bendall, M.H. Spitzer, G.P. Nolan, K. Kobayashi, et al. Immunological Gascoigne, N.R., V. Rybakin, O. Acuto, and J. Brzostek. 2016. TCR Signal Genome Consortium. 2013. The transcriptional landscape of αβ T cell Strength and T Cell Development. Annu. Rev. Cell Dev. Biol. 32:327–348. differentiation. Nat. Immunol. 14:619–632. https://doi.org/10.1038/ni https://doi.org/10.1146/annurev-cellbio-111315-125324 .2590 Glusman, G., L. Rowen, I. Lee, C. Boysen, J.C. Roach, A.F. Smit, K. Wang, B.F. Mombaerts, P., J. Iacomini, R.S. Johnson, K. Herrup, S. Tonegawa, and V.E. Koop, and L. Hood. 2001. Comparative genomics of the human and Papaioannou. 1992. RAG-1-deficient mice have no mature B and T mouse T cell receptor loci. Immunity. 15:337–349. https://doi.org/10 lymphocytes. Cell. 68:869–877. https://doi.org/10.1016/0092-8674(92) .1016/S1074-7613(01)00200-X 90030-G Hsieh, C.S., H.M. Lee, and C.W. Lio. 2012. Selection of regulatory T cells in the Moran, A.E., K.L. Holzapfel, Y. Xing, N.R. Cunningham, J.S. Maltzman, J. thymus. Nat. Rev. Immunol. 12:157–167. https://doi.org/10.1038/nri3155 Punt, and K.A. Hogquist. 2011. T cell receptor signal strength in Treg Huo, J., S. Xu, and K.P. Lam. 2010. Fas apoptosis inhibitory molecule regulates and iNKT cell development demonstrated by a novel fluorescent re- T cell receptor-mediated apoptosis of thymocytes by modulating Akt porter mouse. J. Exp. Med. 208:1279–1289. https://doi.org/10.1084/jem activation and Nur77 expression. J. Biol. Chem. 285:11827–11835. https:// .20110308 doi.org/10.1074/jbc.M109.072744 Na, S.Y., A. Patra, Y. Scheuring, A. Marx, M. Tolaini, D. Kioussis, B.A. Huseby, E.S., J. White, F. Crawford, T. Vass, D. Becker, C. Pinilla, P. Marrack, Hemmings, T. Hünig, and U. Bommhardt. 2003. Constitutively active and J.W. Kappler. 2005. How the T cell repertoire becomes peptide and protein kinase B enhances Lck and Erk activities and influences thy- MHC specific. Cell. 122:247–260. https://doi.org/10.1016/j.cell.2005.05 mocyte selection and activation. J. Immunol. 171:1285–1296. https://doi .013 .org/10.4049/jimmunol.171.3.1285 Iborra, S., M. Soto, L. Stark-Aroeira, E. Castellano, B. Alarcón, C. Alonso, E. Paster, W., A.M. Bruger, K. Katsch, C. Grégoire, R. Roncagalli, G. Fu, N.R. Santos, and E. Fernández-Malavé. 2011. H-ras and N-ras are dispensable Gascoigne, K. Nika, A. Cohnen, S.M. Feller, et al. 2015. A THEMIS:SHP1 for T-cell development and activation but critical for protective Th1 complex promotes T-cell survival. EMBO J. 34:393–409. https://doi.org/ immunity. Blood. 117:5102–5111. https://doi.org/10.1182/blood-2010-10 10.15252/embj.201387725 -315770 Price, D.A., J.M. Brenchley, L.E. Ruff, M.R. Betts, B.J. Hill, M. Roederer, R.A. Juntilla, M.M., and G.A. Koretzky. 2008. Critical roles of the PI3K/Akt sig- Koup, S.A. Migueles, E. Gostick, L. Wooldridge, et al. 2005. Avidity for naling pathway in T cell development. Immunol. Lett. 116:104–110. antigen shapes clonal dominance in CD8+ T cell populations specific for https://doi.org/10.1016/j.imlet.2007.12.008 persistent DNA viruses. J. Exp. Med. 202:1349–1361. https://doi.org/10 Kaye, J., M.L. Hsu, M.E. Sauron, S.C. Jameson, N.R. Gascoigne, and S.M. .1084/jem.20051357 Hedrick. 1989. Selective development of CD4+ T cells in transgenic mice Rodrı́guez-Borlado, L., D.F. Barber, C. Hernández, M.A. Rodrı́guez-Marcos, A. expressing a class II MHC-restricted antigen receptor. Nature. 341: Sánchez, E. Hirsch, M. Wymann, C. Martı́nez-A, and A.C. Carrera. 746–749. https://doi.org/10.1038/341746a0 2003. Phosphatidylinositol 3-kinase regulates the CD4/CD8 T cell dif- Kaye, J., N.J. Vasquez, and S.M. Hedrick. 1992. Involvement of the same re- ferentiation ratio. J. Immunol. 170:4475–4482. https://doi.org/10.4049/ gion of the T cell antigen receptor in thymic selection and foreign jimmunol.170.9.4475 peptide recognition. J. Immunol. 148:3342–3353. Rojas, A.M., G. Fuentes, A. Rausell, and A. Valencia. 2012. The Ras protein Kieback, E., E. Hilgenberg, U. Stervbo, V. Lampropoulou, P. Shen, M. Bunse, superfamily: evolutionary tree and role of conserved amino acids. J. Cell Y. Jaimes, P. Boudinot, A. Radbruch, U. Klemm, et al. 2016. Thymus- Biol. 196:189–201. https://doi.org/10.1083/jcb.201103008 Martı́nez-Riaño et al. Journal of Experimental Medicine 2446 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959 Savage, P.A., and M.M. Davis. 2001. A kinetic window constricts the T cell tolerance. Immunity. 14:523–534. https://doi.org/10.1016/S1074-7613(01) receptor repertoire in the thymus. Immunity. 14:243–252. https://doi 00134-0 .org/10.1016/S1074-7613(01)00106-6 Swat, W., V. Montgrain, T.A. Doggett, J. Douangpanya, K. Puri, W. Vermi, and Shugay, M., D.V. Bagaev, M.A. Turchaninova, D.A. Bolotin, O.V. Britanova, T.G. Diacovo. 2006. Essential role of PI3Kdelta and PI3Kgamma in E.V. Putintseva, M.V. Pogorelyy, V.I. Nazarov, I.V. Zvyagin, V.I. Kirgi- thymocyte survival. Blood. 107:2415–2422. https://doi.org/10.1182/blood zova, et al. 2015. VDJtools: unifying post-analysis of T cell receptor -2005-08-3300 repertoires. PLOS Comput. Biol. 11:e1004503. https://doi.org/10.1371/ Teixeiro, E., and M.A. Daniels. 2010. ERK and cell death: ERK location and journal.pcbi.1004503 T cell selection. FEBS J. 277:30–38. https://doi.org/10.1111/j.1742-4658 Simpson, E.H. 1949. Measurement of diversity. Nature. 163:688. https://doi .2009.07368.x .org/10.1038/163688a0 Vrisekoop, N., J.P. Monteiro, J.N. Mandl, and R.N. Germain. 2014. Revisiting Singer, A., S. Adoro, and J.H. Park. 2008. Lineage fate and intense debate: thymic positive selection and the mature T cell repertoire for antigen. myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat. Immunity. 41:181–190. https://doi.org/10.1016/j.immuni.2014.07.007 Rev. Immunol. 8:788–801. https://doi.org/10.1038/nri2416 Wang, D., M. Zheng, L. Lei, J. Ji, Y. Yao, Y. Qiu, L. Ma, J. Lou, C. Ouyang, X. Starr, T.K., S.C. Jameson, and K.A. Hogquist. 2003. Positive and negative Zhang, et al. 2012. Tespa1 is involved in late thymocyte development selection of T cells. Annu. Rev. Immunol. 21:139–176. https://doi.org/10 through the regulation of TCR-mediated signaling. Nat. Immunol. 13: .1146/annurev.immunol.21.120601.141107 560–568. https://doi.org/10.1038/ni.2301 Sugawara, T., T. Moriguchi, E. Nishida, and Y. Takahama. 1998. Differential Xue, L., L. Chiang, C. Kang, and A. Winoto. 2008. The role of the PI3K-AKT roles of ERK and p38 MAP kinase pathways in positive and negative kinase pathway in T-cell development beyond the beta checkpoint. Eur. selection of T lymphocytes. Immunity. 9:565–574. https://doi.org/10 J. Immunol. 38:3200–3207. https://doi.org/10.1002/eji.200838614 .1016/S1074-7613(00)80639-1 Zamoyska, R., A. Basson, A. Filby, G. Legname, M. Lovatt, and B. Seddon. Suzuki, A., M.T. Yamaguchi, T. Ohteki, T. Sasaki, T. Kaisho, Y. Kimura, R. 2003. The influence of the src-family kinases, Lck and Fyn, on T cell Yoshida, A. Wakeham, T. Higuchi, M. Fukumoto, et al. 2001. T cell- differentiation, survival and activation. Immunol. Rev. 191:107–118. specific loss of Pten leads to defects in central and peripheral https://doi.org/10.1034/j.1600-065X.2003.00015.x Martı́nez-Riaño et al. Journal of Experimental Medicine 2447 RRAS2 sets thymic selection thresholds https://doi.org/10.1084/jem.20181959