Modifying T cells with altered TCR signal proofreading

To elucidate the consequences of disrupting a single bona fide kinetic proofreading step in an otherwise intact biological system, we utilized CRISPR–Cas9 technology to introduce a mutation into the endogenous mouse Lat locus that would be transcribed as the Gly135Asp alteration (Extended Data Fig. 1a–d). In LATG135D mice, the kinetics of a single proofreading signaling step (that is, phosphorylation of LAT Y136, which influences PLC-γ1 recruitment, phosphorylation and activation) is accelerated, endowing T cells with the ability to respond to weak or self-ligands in vitro (Extended Data Fig. 1b).

Expression of G135D LAT alters thymocyte development

Immature thymocytes require TCR signals of appropriate strength to complete thymic development. Since the thymic selection thresholds are differentially regulated in neonates and adults24, we analyzed polyclonal thymocyte development in LATG135D knockin mice or wild-type mice at the neonatal (10- to 14-day-old) and adult (6-week-old) stages.

During the postnatal period, thymus size progressively increased in the wild-type mice, almost doubling between the neonatal and adult stages. In LATG135D mice, the thymus was already enlarged at 2 weeks (Extended Data Fig. 2a). We observed less impact on the double-negative population (Extended Data Fig. 2b) but observed an effect on the double-positive (DP) and single-positive populations in LATG135D mice (Fig. 1a–c and Extended Data Fig. 2c). In adult LATG135D knockin mice, there were approximately 60% fewer single-positive CD4 (CD4SP) and single-positive CD8 (CD8SP) cells than in their wild-type littermates (Fig. 1a–c). Whereas the percentages of neonatal single-positive cells were also lower in LATG135D mice (Fig. 1a–c), the absolute numbers remained comparable to those in neonate wild-type littermates (Fig. 1c). Notably, among the LATG135D single-positive cells, the mature CD62L+H-2Kb+ thymocytes ready for thymic egress were the most substantially affected population (Fig. 1d–f and Extended Data Fig. 2d) in mice of all ages.

Fig. 1: LATG135D affects thymopoiesis and decreases the production of single-positive thymocytes.

a–i, Cellularity of thymi harvested from wild-type (WT) or LATG135D C57BL/6 mice as neonates (2 weeks old) or as adults (6 weeks old). The data are representative of at least four independent experiments. wk, weeks. a, Representative pseudocolor plots depicting the expression of CD4 and CD8. b,c, Bar graphs summarizing the percentages (b) and absolute numbers (c) of CD4SP (left) and CD8SP cells (right) among live thymocytes. d, Expression of the thymocyte maturation markers CD62L and MHC-I H-2Kb on CD4SP cells, including semi-mature (CD62L–H-2Kb–; SM), mature stage 1 (CD62L–H-2Kb+; M1) and mature stage 2 cells (CD62L+H-2Kb+; M2). e,f, Bar graphs summarizing the percentages (e) and absolute numbers (f) of CD62L+H-2Kb+ M2 CD4SP (left) and M2 CD8SP thymocytes (right). g, Representative pseudocolor plots showing CD69 and TCRβ expression profiles of DP thymocytes, including preselection DP1 (CD69–TCRβ–), midselection DP2 (CD69medTCRβmed) and postselection DP3 cells (CD69hiTCRβhi). h,i, Bar graphs summarizing the percentages (h) and absolute numbers (i) of DP2 (left) and DP3 cells (right) among DP thymocytes. In b, c, e, f, h and i, each dot represents an individual mouse (n = 15). In b, **P = 0.0041 and ****P < 0.0001. In c, ****P < 0.0001, NS (not significant) = 0.5393 (left) and NS = 0.2169 (right). In e, ***P = 0.0001 and ****P < 0.0001. In f, ****P < 0.0001, **P = 0.0057 and *P = 0.0295. In h, **P = 0.0012 and ****P < 0.0001. In i, ****P < 0.0001, *P = 0.0209 and NS = 0.6236. Statistical significance was determined by two-tailed Mann–Whitney U-test.

Source data

To further investigate how altering the LAT Y136 kinetic proofreading step affected thymocyte development, we analyzed the DP thymocyte populations. DP cells gradually upregulate the expression of the TCR and the activation marker CD69 upon receipt of selecting signals, progressing from preselection DP1 (CD69–TCR–) to midselection DP2 (CD69medTCRmed) to postselection DP3 (CD69hiTCRhi) thymocytes. The expression of LATG135D resulted in significantly lower frequencies and absolute numbers of DP3 cells in adult mice (Fig. 1g–i), which suggests that the Gly135Asp-induced defects in the CD4SP and CD8SP populations occurred at the DP2-to-DP3 thymocyte transition. Taken together, the data reveal that the expression of LATG135D resulted in substantially smaller CD4SP and CD8SP thymocyte populations in LATG135D mice and that immature adult thymocytes are more sensitive to LATG135D-promoted signaling than neonatal cells.

LATG135D expression triggers negative selection

To establish the cause of the smaller single-positive populations as defective positive selection, disrupted negative selection or death by neglect, we characterized the modifications in TCR signaling conferred by the Gly135Asp alteration. LATG135D or wild-type preselection CD53– thymocytes were isolated ex vivo and labeled with different dilutions of CellTrace Violet (Extended Data Fig. 3a). The cells were mixed and then stimulated with crosslinking anti-CD3ε antibodies. LATG135D preselection cells exhibited a more rapid and much larger increase in cytoplasmic free calcium than that observed in wild-type preselection cells (Fig. 2a and Extended Data Fig. 3b). In contrast, wild-type preselection DP cells showed a slower, more sustained calcium increase (Fig. 2a and Extended Data Fig. 3b). Immunoblot analysis of such ex vivo-stimulated thymocytes further demonstrated that the expression of LATG135D led to enhanced phosphorylation of LAT Y136 and PLC-γ1 in preselection CD53– thymocytes (Fig. 2b and Extended Data Fig. 3c). Importantly, activation of the kinase Zap-70 (as evidenced by phosphorylation of Y493 in its activation loop) and phosphorylation of other LAT tyrosine residues (such as Y195) in LATG135D thymocytes were comparable to levels in wild-type thymocytes (Fig. 2b and Extended Data Fig. 3d). Similar results were observed in peripheral naive CD4 T cells (Extended Data Fig. 3e,f). These results suggest that the Gly135Asp alteration selectively increases the phosphorylation speed and magnitude of Y136 and PLC-γ1.

Fig. 2: LATG135D promotes negative selection in the medulla.

a, Representative calcium traces of wild-type and LATG135D CD53– preselection DP thymocytes were analyzed before and after the addition of streptavidin (SA) to crosslink anti-CD3ε. b, Immunoblot analysis of specific proximal signaling proteins of wild-type or LATG135D CD53– preselection DP thymocytes after crosslinking with anti-CD3ε antibody. MW, molecular weight of protein ladders (kDa). The two columns of labels on the left represent the protein name and the amino acid residue, respectively. The ‘p-’ indicates phosphorylation. c, Histogram of the expression of eGFP in various thymocyte developmental subsets from wild-type and LATG135D Nur77–eGFP reporter mice. d, Pseudocolor plots of the expression of CCR7 and cleaved caspase-3 (aCasp3) in DP thymocytes. e, Bar graphs summarizing the percentages of DP thymocytes undergoing apoptosis in the cortex (CCR7–aCasp3+) or ready to migrate to the medulla (CCR7+aCasp3+). Each dot represents a single mouse (n = 11). ****P < 0.0001 and NS = 0.5726. f, Immunoblot analysis of the total protein expression of Bim and aCasp3 of sorted wild-type and LATG135D DP thymocytes. g,h, Analysis of the expression of CD5 and aCasp3 on total thymocytes (CD5+TCRβ+; an example of the gating strategy is shown in Extended Data Fig. 3e). Representative pseudocolor plots (g) and summarized bar graphs (h) are shown. The numbers in g show the percentages of apoptotic (aCasp3+) and nonapoptotic (aCasp3–) cells. Each symbol in h represents a single mouse (n = 10). ****P < 0.0001. i–k, Representative pseudocolor plots (i) showing the expression of CCR9 and CCR7 on CD5+TCRβ+aCasp3+ thymocytes. The bar graphs show the percentages of CCR7– and CCR7+ CD5+TCRβ+aCasp3+ thymocytes, indicative of clonal deletion in the cortex (CCR7–) versus cells ready to migrate to the medulla (CCR7+) (j) and stage of development (CCR9+CCR7–, CCR9+CCR7+ or CCR9–CCR7–; k). Each dot represents an individual mouse (n = 10). **P = 0.0011, ***P = 0.0001, ****P < 0.0001, NS = 0.0892 (j, left), NS = 0.1230 (j, right) and NS = 0.3930 (k). In a–k, the data are representative of two (f), three (a,c,i–k) or four (b,d,e,g,h) independent experiments. Statistical significance was determined by two-tailed Mann–Whitney U-test.

Source data

Next, we investigated TCR signaling in LATG135D mice following physiologically relevant positive selection stimulation in the thymus25,26,27. Consistent with the in vitro biochemical findings (Fig. 2b), the LATG135D T cells demonstrated stronger orphan nuclear hormone receptor Nur77 activation in vivo (Fig. 2c), probably due to encountered self-pMHCs, as indicated by flow cytometric analysis of LATG135D Nur77–enhanced green fluorescent protein (eGFP) reporter bacterial artificial chromosome transgenic mice28. Notably, there were more eGFP+ cells among post-DP2 LATG135D thymocytes than among corresponding cells from wild-type littermates (Fig. 2c and Extended Data Fig. 4a) and the geometric mean fluorescence intensity of eGFP was also higher in LATG135D thymocytes than in wild-type cells (Fig. 2c and Extended Data Fig. 4b). It was particularly noteworthy that twice as many LATG135D compared with wild-type DP cells displayed a cleaved caspase-3+ (aCasp3+) and chemokine receptor CCR7+ phenotype, consistent with ongoing apoptosis due to clonal deletion of thymocytes migrating to the medulla25,29,30,31 (Fig. 2d,e). Immunoblot analysis of both adult and neonatal LATG135D DP thymocytes compared with wild-type counterparts further confirmed elevated expression of aCasp3 and proapoptotic Bcl-2 family member Bim (Fig. 2f).

Since clonal deletion can occur throughout the maturation process, we furthered assessed the clonal deletion of total thymocytes that received TCR signals31 (Extended Data Fig. 4c). Among thymocytes that had experienced TCR signals based on CD5 upregulation31 (Extended Data Fig. 4d), there was a threefold increase in the aCasp3+ population in the adult LATG135D mice compared with that in wild-type littermates (Fig. 2g,h) and an eightfold increase in LATG135D neonates (Extended Data Fig. 4e,f). We measured the expression levels of CCR9 and CCR7 on TCR-signaled aCasp3+ thymocytes to approximate the effects of the alteration on the anatomic location and timing of clonal deletion. For both wild-type and LATG135D thymocytes, roughly 60% of clonal deletion occurred in the cortex (Fig. 2i,j), consistent with previous reports25,31,32. Interestingly, in comparison with wild-type thymocytes, a larger proportion of LATG135D thymocytes underwent clonal deletion at the semi-mature, proliferation-incompetent CCR9+ stage (Fig. 2i,k), which may explain the altered maturation pattern in LATG135D mice (Fig. 1c,d). In addition, clonal deletion during CD4SP maturation usually correlates with failed regulatory T-cell (Treg cell) development1. We observed fewer Treg cells in LATG135D mice (Extended Data Fig. 4g,h). These results indicate that the expression of LATG135D promotes negative selection, possibly due to enhanced thymocyte reactivity to self-pMHC stimuli caused by augmented TCR-dependent LAT Y136–PLC-γ1 signaling.

LATG135D promotes homeostatic proliferation

To investigate how T cells with altered kinetic proofreading and potentially enhanced self-reactivity respond in the periphery, we examined the phenotypic and functional characteristics of polyclonal peripheral LATG135D CD4 and CD8 splenocytes. LATG135D mice had fewer CD8 T cells (Extended Data Fig. 5a) than their wild-type littermates, whereas LATG135D-expressing CD4 T cells were relatively less affected (Extended Data Fig. 5b). Nonetheless, both LATG135D CD4 and CD8 populations included enlarged CD62L–CD44+ populations that were age dependent and obvious only in adult mice (Fig. 3a–d and Extended Data Fig. 5c,d). Interestingly, the LATG135D mice also harbored a substantial population of CD8 T cells that adopted a central memory-like phenotype (Fig. 3c,e and Extended Data Fig. 5e)—a population that is driven by higher self-reactivity33,34 and exhibited enhanced responsiveness to lower-dose anti-CD3 stimulation (Extended Data Fig. 5f). Similar phenotypes were also observed in lymph nodes (Extended Data Fig. 5g,h).

Fig. 3: LATG135D augments self-peptide-driven homeostatic proliferation of peripheral T cells.

a,c, Representative pseudocolor plots of the expression of CD62L and CD44 on peripheral spleen CD4 (a) and CD8 T cells (c) from wild-type versus LATG135D neonatal (2 week) and adult (6 week) mice. The numbers associated with the gates show the percentages of naive (CD62L+CD44–), central memory (CD62L+CD44+) and effector memory (CD62L–CD44+) cells. b,d,e, Bar graphs depicting the percentages of effector memory (CD62L–CD44+) cells among peripheral CD4 (b) and CD8 (d) T cells and the percentages of central memory (CD62L+CD44+) cells among peripheral CD8 T cells (e). Each dot represents a single mouse (n = 15). The data are representative of at least five independent experiments. ****P < 0.0001, NS = 0.4302 (b) and NS = 0.9588 (d). f–i, Naive CD4 (f,g) or CD8 (h,i) T cells were sorted from 4- to 5-week-old wild-type or LATG135D mice, labeled with CellTrace Violet and adoptively transferred intravenously into congenic hosts (CD45.1+), MHC-II–/– hosts or Tap1–/–B2m–/– hosts (as indicated) that had been sublethally irradiated (300 rads) the day before. The dilution of CellTrace Violet was assessed by flow cytometry 4 d post-transfer. f,h, Representative flow plots of CellTrace Violet dilution and the expression of CD5. g,i, Bar graphs summarizing the percentages of adoptively transferred CD4 (g) and CD8 cells (i) that underwent proliferation. Each dot represents an individual mouse (n = 6 for the CD45.1+ C57BL/6 host and n = 4 for the MHC-II–/– and Tap1–/–B2m–/– hosts. The data were compiled from three independent experiments. **P = 0.0043 (g), **P = 0.0022 (i), NS = 0.3429 (g) and NS = 0.9429 (i). Statistical significance in b, d, e, g and i was determined by two-tailed Mann–Whitney U-test.

Source data

To further investigate the mechanisms behind the altered cellularity in the periphery of LATG135D mice, we adoptively transferred sorted naive LATG135D CD4 T cells labeled with CellTrace Violet proliferation dye into sublethally irradiated congenic CD45.1+ C57BL/6 hosts to examine the homeostatic proliferation. After 4 days, we observed that LATG135D CD4 T cells proliferated more robustly than wild-type CD4 T cells (Fig. 3f,g and Extended Data Fig. 5i); approximately 80% of LATG135D CD4 T cells underwent proliferation compared with 49% of wild-type CD4 T cells (Fig. 3g). Naive LATG135D CD8 T cells displayed similarly stronger proliferation than wild-type CD8 T cells (Fig. 3h,i and Extended Data Fig. 5j). Notably, transfer into sublethally irradiated MHC-II–/– hosts, which prevents interaction with pMHC-II, rendered LATG135D CD4 T cells nonproliferative (Fig. 3f,g). Restricting the repertoire of MHC-I-bound self-peptides by employing sublethally irradiated Tap1–/–B2m–/– hosts revealed similar self-pMHC-driven homeostatic proliferation of LATG135D CD8 T cells (Fig. 3h,i). These data show that LATG135D T cells exhibit enhanced reactivity/responsiveness to self-pMHCs, which contributes to their greater homeostatic proliferation potential.

LATG135D T cells exhibit hyper-responsiveness to self-ligands

To more thoroughly study the effects of LATG135D on self-pMHC reactivity, we introduced the LATG135D mutation onto the OT-I TCR transgenic Rag1–/– background. LATG135D.OT-I.Rag1–/– mice exhibited phenotypes consistent with those observed in polyclonal C57BL/6 mice, including a smaller CD8SP population (Extended Data Fig. 6a,b) and enhanced negative selection (Extended Data Fig. 6c,d) in the thymus, as well as augmented CD44+ populations in the periphery (Extended Data Fig. 6e–g). CD5 expression was also elevated in LATG135D.OT-I.Rag1–/– CD8 T cells, while the expression levels of OT-I TCR (Vα2), CD3 and CD28 were comparable to those of wild-type T cells (Extended Data Fig. 6h). Similar phenotypes resulting from LATG135D alteration were also observed on OT-II.Rag1–/–, SMARTA.Rag1–/– and AND.Rag1–/– TCR transgenic backgrounds (Extended Data Fig. 7a–f).

To further test whether the expression of LATG135D regulates T-cell ligand discrimination, we utilized four altered peptide ligands (APLs) and two self-peptides, Catnb and Cappa1, that are recognized by the OT-I TCR35. Using in vitro fetal thymic organ cultures (FTOCs)36,37, we observed that significantly fewer CD8SP cells developed in LATG135D.OT-I.Rag1–/–.Tap1–/– cultures than in wild-type LAT cultures treated with the agonist ovalbumin (OVA), or partial agonists Q4R7 or T4 (Fig. 4a and Extended Data Fig. 7g). In contrast, treatment with the weak agonists V4 and G4 or the self-peptide Catnb promoted stronger positive selection of LATG135D.OT-I.Rag1–/–.Tap1–/– thymocytes, as indicated by a roughly twofold increase in CD8SP cells compared with those in wild-type LAT cell cultures (Fig. 4a and Extended Data Fig. 7g). Further analysis of OVA APL two-dimensional (2D) affinity (Extended Data Fig. 7h,i), along with the frequency of CD8SP, showed that the expression of LATG135D converts the borderline negative selectors (for example, T4 and Q4H7) into pure negative selectors and augments the selection efficiency of positive selectors (for example, V4, G4, Catnb and Cappa1) (Extended Data Fig. 7j).

Fig. 4: LATG135D promotes OT-I CD8 T-cell effector function and augments sensitivity to weak ligand stimuli.

a, Fetal thymi from wild-type or LATG135D.OT-I.Rag1–/–.Tap1–/– mice were cultured with OVA peptide, OVA APLs or self-peptides, as indicated. The percentages of CD8SP cells were analyzed on day 4. The data are representative of two independent experiments. Representative flow plots show the development of CD8SP cells in FTOC. b, Naive wild-type or LATG135D.OT-I.Rag1–/– TCR transgenic CD8 T cells were sorted from 4- to 5-week-old mice and stimulated overnight with TCRα–/– antigen-presenting cells pulsed with OVA peptide, OVA APLs or self-peptide Catnb or Cappa1 over a wide range of peptide concentrations (as indicated on the x axis). The upregulation of CD69 was analyzed the next day by flow cytometry. CD69+ cells were plotted against peptide concentrations. The data represent means ± s.d. (n = 3 independent experiments). c, Naive wild-type or LATG135D.OT-I.Rag1–/– TCR transgenic CD8 T cells were sorted from 4- to 5-week-old mice, labeled with CellTrace Violet and cocultured with TCRα–/– antigen-presenting cells pulsed with OVA, APLs (V4 or G4), self-peptide (Catnb or Cappa1) or unrelated peptide (VSV). The fluorescence profile of CellTrace Violet and expression of CD5 were assessed on day 4. The data are representative of at least three independent experiments. d, Representative flow plots depicting the production of the cytokines TNF and IFNγ by naive cells from wild-type or LATG135D.OT-I.Rag1–/– mice stimulated with OVA-, V4- or Catnb-pulsed TCRα–/– antigen-presenting cells overnight. The data are representative of three independent experiments. e, Naive cells from wild-type or LATG135D.OT-I.Rag1–/– mice were sorted from 4- to 5-week-old mice and stimulated with OVA-, V4- or Catnb-pulsed TCRα–/– antigen-presenting cells overnight. The production of IL-2 and expression of pSTAT5 were measured by intracellular staining and flow cytometry. The data are representative of four independent experiments.

Source data

Next, we isolated naive LATG135D or wild-type LAT.OT-I.Rag1–/– peripheral CD8 T cells from 4- to 5-week-old mice (Extended Data Fig. 8a), stimulated the cells with OVA- or APL-pulsed antigen-presenting cells and examined the upregulation of CD69 (Fig. 4b). Whereas LATG135D.OT-I.Rag1–/– CD8 T cells responded only slightly more sensitively than wild-type OT-I.Rag1–/– CD8 T cells to OVA or the partial agonists Q4R7, T4 and Q4H7, they responded with substantially greater sensitivity to the weak ligands V4 and G4 and self-peptides Catnb and Cappa1 (Fig. 4b). Plotting the potency by 2D (Extended Data Figs. 8b) or 3D (Extended Data Fig. 8c) affinity revealed that the expression of LATG135D lowers the TCR discriminatory power (flattening the slope on the log–log plot)4, particularly in response to the weak ligands and self-peptides.

These weak ligands or self-peptides also promoted robust proliferative responses by naive LATG135D.OT-I.Rag1–/– CD8 T cells in contrast with wild-type cells, as revealed by the dilution of CellTrace Violet dye (Fig. 4c and Extended Data Fig. 8d). Similarly, after culture with OVA- or APL-pulsed antigen-presenting cells, a significantly greater number of LATG135D compared with wild-type LAT.OT-I.Rag1–/– CD8 T cells were Ki-67+ cells the next day (Extended Data Fig. 8e,f). These Ki-67+ cells also exhibited upregulation of endogenous Nur77 (Extended Data Fig. 8f), which is evidence for TCR recognition-driven proliferation. In addition, the weak ligand V4 and self-peptide Catnb induced more LATG135D versus wild-type LAT.OT-I.Rag1–/– CD8 T cells to produce the cytokines interferon-γ (IFNγ) and tumor necrosis factor (TNF) (Fig. 4d). Interestingly, the expression of LATG135D had the opposite effect on the production of interleukin-2 (IL-2) (Fig. 4e). Next, we generated cytotoxic T lymphocytes (CTLs) and found that LATG135D.OT-I.Rag1–/– CTLs mediated greater cytotoxicity against APL-pulsed EL4 cells at lower CTL-to-EL4 ratios than wild-type LAT.OT-I.Rag1–/– CTLs (Extended Data Fig. 8g). Weaker ligands or self-peptides were also able to activate LATG135D CD4 T cells expressing OT-II, SMARTA or AND TCRs (Extended Data Fig. 8h–j) to a greater degree than wild-type LAT CD4 T cells. These results suggest that the expression of LATG135D may enable T cells to adopt a stronger effector cell program when challenged with weaker pMHCs or even self-pMHCs, suggesting that LATG135D OT-I cells are less able to discriminate a true agonist from a weak agonist or even a self-pMHC.

LATG135D facilitates the nuclear translocation of NFAT

To determine how the altered LAT Y136-centric kinetic proofreading step modulates the activation of specific transcription factors that are responsive to distinct signaling pathways, we examined the activation of transcription factors in isolated cell nuclei38 from naive wild-type or LATG135D.OT-I.Rag1–/– CD8 T cells (Fig. 5). We first characterized nuclear NFAT1, which translocates from the cytoplasm to the nucleus following its dephosphorylation by the calcium–calmodulin-activated phosphatase calcineurin, the consequence of direct LAT–PLC-γ1–calcium downstream signaling. OVA stimulation induced rapid nuclear localization of NFAT1, and the expression of LATG135D substantially promoted increased accumulation of NFAT1 in nuclei (Fig. 5a and Extended Data Fig. 8k). At none of the responses of the wild-type cells did NFAT translocation equal that of the LATG135D variant. NFAT signaling is necessary for the induction of transcripts of Nur77 (ref. 39), and the magnitude of the nuclear expression of Nur77 was also greatly enhanced in LATG135D.OT-I.Rag1–/– CD8 T cells (Fig. 5b). Interestingly, we did not observe substantial differences in nuclear translocation of nuclear factor-κB (NF-κB) (Fig. 5c) or Egr-2 (Fig. 5d) between LATG135D and wild-type LAT.OT-I CD8 T cells, which are regulated through costimulatory signaling in addition to TCR signals40,41. These data suggest that LATG135D-promoted PLC-γ1 and calcium signals enhance the nuclear translocation of NFAT1 and transcriptional induction of Nur77, which is highly sensitive to NFAT, both of which may contribute to the hyper-responsiveness of LATG135D T cells.

Fig. 5: LATG135D-mediated signaling promotes NFAT1 and Nur77 translocation into the nucleus.

a–d, Naive wild-type or LATG135D.OT-I.Rag1–/– CD8 T cells from 4- to 5-week-old mice were sorted and subjected to nuclear staining with CellTrace Blue dyes, then stimulated in vitro with 10 or 0.1 nM OVA peptide-pulsed TCRα–/– splenocytes over a time course of 180 or 240 min (as indicated on x axis). Cell nuclei were isolated according to a published protocol, fixed and permeabilized, then subjected to antibody staining for NFAT1, Nur77, NF-κB or Egr-2. Nuclear NFAT1 (a), Nur77 (b), NF-κB (c) and Egr-2 (d) expression was analyzed by flow cytometry. The percentage of positive nuclei (for NFAT1, NF-κB and Egr-2) or mean fluorescence intensity (MFI; Nur77) for individual conditions was plotted against the stimulation time to depict the nuclear translocation kinetics of transcription factors, as indicated. The data represent means ± s.d. (n = 4 independent experiments).

Source data

G135D LAT augments T-cell expansion to Listeria in vivo

To examine how these LATG135D T cells balance tolerance and immune responsiveness, we used an immune challenge model. We adoptively transferred sorted CD62L+CD44– naive CD45.2+ LATG135D or wild-type LAT.OT-I.Rag1–/– spleen CD8 T cells into congenic CD45.1+ hosts (Extended Data Fig. 9a) and infected the mice with recombinant Listeria monocytogenes strains engineered to express OVA (Lm-OVA) or very weak APL V4 (Lm-V4) the next day35. On day 7 postinfection, LATG135D.OT-I.Rag1–/– CD8 T cells consistently expanded to a greater degree than wild-type OT-I.Rag1–/– CD8 T cells in the Lm-V4 infection settings (Fig. 6a,b). Notably, OT-I TCR affinity to the V4 peptide is reported to be substantially weaker, within the range of characterized positively selecting APLs5,35. Lm-V4 infection resulted in the activation of only ~0.03% of wild-type T cells, but led to expansion of 0.15% of LATG135D T cells (Fig. 6b). Interestingly, OT-I T cells expressing LATG135D and wild-type OT-I T cells responded comparably to Lm-OVA, and activated LATG135D.OT-I.Rag1–/– CD8 T cells showed comparable cytokine production capacity to their wild-type counterparts (Extended Data Fig. 9b,c). These results are consistent with our in vitro data showing that modification of a kinetic proofreading step has a greater effect on weak ligand stimulation. In addition, infection with Lm-OVA emphasized the shift in effector versus memory cell fate decisions. We observed that the KLRG1–CD127+ memory precursor population was substantially decreased by more than twofold among transferred LATG135D.OT-I.Rag1–/– CD8 T cells compared with transferred LAT wild-type cells in response to Lm-OVA infection (Fig. 6c,d and Extended Data Fig. 9d), whereas the short-lived KLRG1+CD127– effector cell population was consistently larger.

Fig. 6: LATG135D augments the CD8 T-cell response in vivo.

a, Representative pseudocolor plots showing the frequency of CD45.2 OT-I T cells among total CD8 T cells on day 7 postinfection of L. monocytogenes expressing OVA (Lm-OVA) or V4 (Lm-V4). b, Bar graphs depicting the frequency of OT-I T cells among total CD8 T cells on day 7 postinfection (n = 4). *P = 0.0286 and NS = 0.2. c, Representative pseudocolor plots of the expression of KLRG1 and CD127 on wild-type or LATG135D.OT-I.Rag1–/– CD8 T cells on day 7 postinfection.