To determine translational dynamics in developing thymocytes, we injected wildtype C57BL/6 (WT) mice with O-propargyl puromycin (OP-Puro), a cell-permeable puromycin analog that is incorporated into newly synthesized proteins as a measure of protein synthesis rates (Hidalgo San Jose and Signer, 2019; Tong et al., 2020). Animals were euthanized 1 hr after OP-Puro injection, and thymocyte subsets (gated as shown in Figure 1—figure supplement 1B,C) were assessed by flow cytometry for OP-Puro incorporation (Figure 1A). Compared to DP and mature single positive thymocytes, DN2 to DN4 thymocytes incorporated the most OP-Puro (Figure 1B), a likely reflection of their high metabolic and proliferative activity (Carpenter and Bosselut, 2010; Kreslavsky et al., 2012; Nagelreiter et al., 2018). To assess the relationship between translational activity and proteome quality, we stained WT thymocytes with tetraphenylethene maleimide (TMI), a cell-permeable reagent that only fluoresces when bound to free thiol groups typically exposed on misfolded or unfolded proteins (Chen et al., 2017; Hidalgo San Jose et al., 2020). Intriguingly, despite comparable and high protein synthesis rates across DN2 to DN4 thymocytes, we found that DN4 thymocytes displayed markedly lower levels of misfolded/unfolded proteins (Figure 1C). These observations suggested that the DN3-to-DN4 transition, which is initiated by β-selection, is accompanied by induction of proteome quality control mechanisms.

To understand the protein quality control mechanisms operating in thymocytes, we performed quantitative PCR to determine expression of genes encoding ER protein quality control machinery. We found elevated expression of the core ERAD (Sel1l) and the UPR (Xbp1, Ddit3 (Chop), Atf4, Dnajb9, and Hspa5 (Bip)) genes in DN3 thymocytes (Figure 1D). Notably, induction of these genes peaked in the DN3 thymocyte stage in which β-selection is initiated (Takahama, 2006) and preceded the reduction of misfolded/unfolded proteins in DN4 cells. These results prompted us to hypothesize and explore whether β-selection signals induce proteome quality control mechanisms in DN3 cells to enable subsequent stages of thymocyte development.

To resolve the ER proteostasis machinery required for the development of thymocytes, we conditionally deleted individual genes encoding key mediators of the UPR (Xbp1, Perk) and ERAD (Sel1l) using the hCD2-iCre transgene (Siegemund et al., 2015). In this model, significant Cre activity initiated in ETP thymocytes (Shi and Petrie, 2012; Siegemund et al., 2015) and was efficient in depleting genes in subsequent stages of thymocyte development and mature T cells (Figure 2—figure supplement 1A,B). Deletion of the UPR mediator Xbp1 or Perk had no effect on T cell development as thymic cellularity, numbers of DN, DP, single positive (SP) thymocytes and splenic T cell numbers were comparable in gene-deficient and littermate control animals (Figure 2—figure supplement 1C–J). Similarly, Vav1-iCre-mediated deletion of Atf6a which initiates in bone marrow hematopoietic cell progenitors (Joseph et al., 2013) did not perturb thymocyte development (Figure 2—figure supplement 1K–N).

Strikingly, CD2-iCre-mediated deletion of the ERAD core component Sel1l (Sel1l^flox/flox; CD2-iCre mice, hereafter designated as Sel1l CKO) resulted in a markedly decreased thymus size and cellularity, with significantly reduced and dispersed medullary regions compared to control littermates (Sel1l^flox/flox; Ctrl) (Figure 2A–D). The reduced thymus cellularity was accompanied by a profound reduction of peripheral T cells in the spleen and lymph nodes from Sel1l CKO mice compared to control animal (Figure 2—figure supplement 1O–R). Sel1l deletion had no impact on γδ T cells (Figure 2—figure supplement 1S), indicating that within the T-cell lineage, SEL1L is selectively required for αβ T cell development.

While Sel1l CKO mice had similar numbers of DN thymocytes (Figure 2E and Figure 2—figure supplement 2A), they showed significantly reduced numbers of DP and mature SP thymocytes compared to littermate controls (Figure 2F). This finding suggests that SEL1L functioned during the DN to DP thymocyte transition. To clarify this possibility, we generated and analyzed thymocyte developmental stages in Sel1l^flox/flox; Cd4-Cre mice. Unlike CD2-iCre which initiated in ETP (Siegemund et al., 2015), Cd4-Cre initiated in immature single-positive (ISP, CD8⁺CD24⁺TCRβ^-) thymocytes (Gegonne et al., 2018; Kadakia et al., 2019; Xu et al., 2016) and only significantly depleted Sel1l in ISPs and later stage thymocytes (Figure 2—figure supplement 2B,C). Sel1l^flox/flox; Cd4-Cre mice exhibited indistinguishable thymic cellularity, immature DN, DP and SP thymocytes numbers from control mice (Figure 2—figure supplement 2D–G). Thus, whereas SEL1L is dispensable for differentiation of post-DN4 thymocytes (i.e. DP, SP, and mature T cells), its expression is critical for DN to DP thymocyte differentiation.

To delineate the DN thymocyte developmental stage at which SEL1L is required, we generated 1:1 mixed bone marrow (BM) chimeras by transplanting equal numbers of whole BM cells from control (Sel1l^flox/flox, CD45.2⁺) or Sel1l CKO (Sel1l^flox/flox; CD2-iCre, CD45.2⁺) mice along with congenic (CD45.1⁺) wild-type (WT) competitor BM cells into irradiated CD45.1⁺ recipient mice (Figure 2G). Fourteen weeks after transplantation, control and Sel1l CKO donors reconstituted similar numbers of all BM hematopoietic progenitors including Lineage^-Sca-1⁺c-Kit⁺ (LSK) cells, hematopoietic stem progenitor cells (HSPC), multipotent progenitor (MPP), and myeloid progenitors (Lineage^-Sca-1^-c-Kit⁺LS-K) cells (Figure 2—figure supplement 2H). We found substantial defective thymus reconstitution starting from the DN4 stage from Sel1l-CKO donors (Figure 2H,I). Sel1l CKO-donor-derived DN3 to DN4 ratio was significantly increased compared to controls (Figure 2—figure supplement 2I), suggesting that Sel1l depletion compromised the fitness of DN3 thymocytes in which β-selection occurs and impaired their transition to the DN4 stage. The impaired DN thymus reconstitution from Sel1l CKO donors was accompanied by severe defects in subsequent donor-derived DP and SP thymocytes (Figure 2H,I), as well as peripheral T cells in the spleen (Figure 2—figure supplement 2J). These data demonstrate a cell-intrinsic requirement of Sel1l for DN3 thymocyte progression to later stages of thymocyte differentiation. Sel1l deletion also caused significant B cell reconstitution defects (Figure 2—figure supplement 2J), consistent with previous publications showing that SEL1L/HRD1 ERAD is required for B cell development through regulating pre-BCR (Ji et al., 2016; Yang et al., 2018).

To further clarify the requirement for SEL1L in DN3-to-DN4 thymocyte transition during β-selection, we used the in vitro T cell differentiation system of culturing immature thymocytes on monolayers of OP9 stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1 cells) supplemented with IL-7 and Flt3 ligand (Balciunaite et al., 2005; Holmes and Zúñiga-Pflücker, 2009; Schmitt et al., 2004; Figure 2J). We cultured DN2 thymocytes from control or Sel1l CKO mice on OP9-DL1 cells and found that Sel1l-deficiency markedly abrogated DN4 thymocyte generation (Figure 2K–M). On day 17 of co-culture, only 31.3% DN3 cells remained in control-derived cells, while more than three-fold DN3 cells (71.6%) were found in Sel1l CKO-derived cells (Figure 2K–M), consistent with a block in DN3 to DN4 thymocyte transition. Since only DN3 thymocytes that have successfully undergone β-selection differentiate to the DN4 and DP thymocyte stage, these results reinforce that cell-intrinsic SEL1L activity is required for post β-selection DN thymocyte development.

Next, we assessed the impact of Sel1l deletion on DN thymocyte proliferation and survival, two key outcomes of successful β-selection (Ciofani and Zúñiga-Pflücker, 2005; Kreslavsky et al., 2012). Although BrdU incorporation was similar in control and Sel1l CKO DN3 thymocytes, we observed more BrdU incorporation in Sel1l CKO DN4 thymocytes than that in control DN4 thymocytes (Figure 3A). In agreement, co-staining for Ki-67 and the DNA dye (DAPI) revealed less Sel1l CKO DN4 thymocytes in G0 phase, and more in G1 and S/G2/M phase (Figure 3B,C). Sel1l CKO DN3 cells showed similar cell cycle kinetics as control DN3 thymocytes, in line with their normal levels of BrdU incorporation (Figure 2—figure supplement 2K,L). The higher proliferation of endogenous Sel1l-deficient DN4 thymocytes may be a compensation for their compromised fitness. This likely explains the paradoxical observation that Sel1l CKO donors generated markedly reduced DN4 thymocytes in BM chimeras (Figure 2H,I), yet Sel1l CKO mice had comparable DN4 thymocytes as control mice in steady state (Figure 2E).

That, despite their higher proliferative status, Sel1l CKO DN4 thymocytes failed to progress to DP thymocytes and generate mature T-cells prompted us to ask if Sel1l deficiency resulted in apoptosis of β-selected thymocytes. To test this possibility, we cultured equal number of DN3 thymocytes from control or Sel1l CKO mice on OP9-DL1 stromal cells and assessed their apoptosis in vitro. Indeed, Sel1l CKO DN3 thymocytes showed higher apoptosis measured by proportions of Annexin-V positive cells (Figure 3D). To confirm that Sel1l-deficiency resulted in DN thymocyte apoptosis in vivo, we histologically enumerated apoptosis in thymic sections by measuring active Caspase-3 which degrades multiple cellular proteins and is responsible for morphological changes and DNA fragmentation in cells during apoptosis (Bai et al., 2013). Compared to controls, Sel1l CKO thymus showed more active Caspase-3 apoptotic cells in the cortex area (Figure 3E,F), which is typically populated by DN thymocytes (Trampont et al., 2010). This observation was further confirmed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining (Figure 3G,H). Taken together, we concluded that Sel1l-deficient β-selected DN3 cells were undergoing apoptosis as they differentiated into DN4 thymocytes.

Having established that the SEL1L-ERAD is a crucial proteostasis machinery required for β-selection, we next sought to understand the thymic signals which regulate Sel1l expression in DN thymocytes. Because high Sell1l expression (Figure 1D) coincided with high levels of Notch1 in DN2 and DN3 thymocytes (Figure 4A), we explored the possibility that Notch ligands might activate the ERAD machinery to enable DN thymocytes to maintain proteostasis during β-selection. Stimulation of the thymoma cell line EL4 with Notch ligand Delta-like 4 (DLL4) (Camelo et al., 2012; Fitzgerald and Greenwald, 1995) induced expression of the genes involved in ERAD including Sel1l, Hrd1, Os9, and Edem1 as well as SEL1L proteins (Figure 4B,C). Induction of these genes by DLL4 was concomitant with the induction of classical Notch targets like Hes1, Deltex1, and Ptcra (pre-Tα) (Figure 4—figure supplement 1A).

To further determine whether Notch regulates expression of ERAD component genes, we treated freshly isolated primary DN3 thymocytes with the highly specific γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) that blocks ligand-induced cleavage of the Notch intracellular domain (NICD), preventing its nuclear translocation and subsequent transactivation of the RBP-J transcription factor at target genes (Chen et al., 2019; De Obaldia et al., 2013; Schmitt et al., 2004; Bray, 2016). Indeed, treatment with DAPT significantly reduced expression of Notch target genes Hes1, Deltex1, and Ptcra (Figure 4—figure supplement 1B) and also reduced Sel1l, Hrd1, Os9, and Edem1 expression in DN3 thymocytes (Figure 4D). These data demonstrate that Notch signals regulate expression of genes constituting the ERAD machinery.

Analysis of the promoters of the core ERAD genes, Sel1l and Hrd1, revealed conserved binding sites for RBP-J (Figure 4E), the DNA binding partner and master transcription factor of the NICD transactivation complex (Castel et al., 2013; Tanigaki and Honjo, 2007). To interrogate how Notch regulates the ERAD gene components, we performed chromatin immunoprecipitation and qPCR of target DNA (ChIP-qPCR) experiments in EL4 cells. DLL4 stimulation of Notch signaling significantly induced NICD and RBP-J bindings at the promoters of Sel1l, Hrd1, Os9, and Edem (Figure 4F–I).

We cloned the Sel1l and Hrd1 promoters containing the RBP-J-binding sites into the pGL3 firefly luciferase reporter and tested the regulation of these promoters by Notch. When co-transfected with promoter luciferase reporters into HEK293T cells, NICD potently induced both Sel1l and Hrd1 promoter activity in a dose-dependent manner (Figure 4J). In agreement, DLL4 stimulation of Notch signaling in EL4 cells also substantially activated Sel1l and Hrd1 promoter activity (Figure 4K,L). Importantly, mutation of the RBP-J-binding sites abolished DLL4-driven induction of Sel1l or Hrd1 luciferase reporter activity (Figure 4K,L). These data indicate that Notch signaling directly regulates expression of SEL1L ERAD machinery. Interestingly, although ERAD is known to regulate surface receptor expression in a substrate-specific manner (van den Boomen and Lehner, 2015; Xu et al., 2020), Sel1l deletion did not affect surface Notch1 protein levels in DN thymocyte populations (Figure 4—figure supplement 1C). Similarly, SEL1L had little impact on the expression of Notch target genes Hes1 and Ptcra in DN3 thymocytes (Figure 4—figure supplement 1D). These results not only suggest that ERAD does not regulate Notch signaling per se but also imply that thymocyte β-selection defects in Sel1l CKO mice were not due to failure in Notch signal transduction.

The Tcrb allele is rearranged in DN2/DN3 thymocytes and the resulting TCRβ protein pairs with pre-Tα and CD3 complex proteins to form the pre-TCR which, together with Notch, transduces β-selection signals and promotes survival, proliferation and further differentiation of DN3 thymocytes (Ciofani et al., 2004; Michie and Zúñiga-Pflücker, 2002; Sambandam et al., 2005). DN3 thymocytes that fail to undergo productive recombination of the Tcrb locus fail β-selection, do not complete the DN4-DP transition and are eliminated by apoptosis (Ciofani et al., 2004). Therefore, to understand how SEL1L regulates β-selection thymocyte survival and the resulting DN-to-DP transition, we first asked whether Sel1l-deficiency caused defective V(D)J recombination. Genomic DNA analysis of Sel1l-deficient DN3 and DN4 thymocytes showed that recombination of Vb5-Jb2, Vb8-Jb2, and Vb11-Jb2 gene segments were not altered (Figure 5—figure supplement 1A), indicating that Sel1l deficiency does not affect Tcrb gene rearrangement.

Next, we asked whether SEL1L regulates the expression and signaling of the pre-TCR complex. Expression of intracellular TCRβ in pre-selected DN3a, post-selected DN3b and DN4 cells was comparable between control and Sel1l CKO mice (Figure 5—figure supplement 1B,C). Sel1l deletion also had no impact on the expression of pre-TCR signaling intermediates including LCK and ZAP-70 in DN3 and DN4 thymocytes (Figure 5—figure supplement 1D).

To further evaluate whether the Sel1l CKO mice phenotype was due to defective pre-TCR signaling, we introduced the MHCII-restricted TCR-β transgene OT-II into Sel1l CKO mice. As previously reported (Kim et al., 2014; Marquis et al., 2014), expression of TCR transgenes like OT-II in early DN thymocytes can rescue β-selection defects caused by defective pre-TCR expression or signaling. However, the OT-II TCR transgene did not rescue T cell development in OT-II. Sel1l CKO mice which showed a >80% decrease in thymic cellularity, DP and SP thymocytes (Figure 5—figure supplement 1E–G). In addition, OT-II TCR failed to rescue impaired DN3 to DN4 thymocyte transition resulting from Sel1l deficiency as DN4 thymocytes were decreased ~60% in OT-II. Sel1l CKO mice compared to littermate controls (Figure 5—figure supplement 1H). Taken together, these data implied that β-selection defects in Sel1l CKO mice were not due to defects in pre-TCR expression or signaling.

To understand the molecular mechanism by which SEL1L ERAD regulates thymocyte survival and differentiation at β-selection, we performed RNA-seq on control and Sel1l CKO DN3 thymocytes. The most upregulated pathways in Sel1l CKO DN3 thymocytes were ER stress response and the UPR, including both IRE1α and PERK pathways (Figure 5A,B). Gene set enrichment analysis (GSEA) also revealed enriched ER stress response in Sel1l CKO thymocytes (Figure 5C–E). These signatures hinted at elevated ER stress in Sel1l CKO DN3 thymocytes. Consistent with elevated ER stress, flow cytometry quantification of ER tracker dye staining indicated significant ER expansion in Sel1l CKO DN3 cells compared to control thymocytes (Figure 5F,G).

To further ascertain the induction of ER stress following Sel1l deletion, we sorted DN3 and DN4 thymocytes from control or Sel1l CKO mice and examined the activation of all three UPR branches. We found a significant increase in IRE1α proteins and in the splicing of its substrate Xbp1 in Sel1l CKO thymocytes (Figure 5H,I and Figure 5—figure supplement 2A,B). We also observed increased PERK and eIF2α phosphorylation as well as increased ATF4 and BIP proteins in Sel1l CKO DN3 thymocytes (Figure 5H). Various ER chaperones, including Calreticulin, Grp94 (Hsp90b1), Hspa5(Bip), Hyou1, and Canx, and other members of ERAD pathway were markedly upregulated in Sel1l CKO DN3 and DN4 thymocytes (Figure 5—figure supplement 2C,D). These data indicate that Sel1l deletion triggers ER stress leading to activation of all three UPR branches in DN thymocytes.

As ERAD alleviates proteotoxic stress by promoting the degradation of misfolded or unfolded proteins, we hypothesized that loss of SEL1L increased proteotoxic stress during β-selection. Indeed, we found that Sel1l CKO DN3 thymocytes exhibited significantly higher staining for misfolded/unfolded proteins with TMI (Figure 5J,K). We also employed proteostat, a molecular rotor dye, to examine protein aggregation in DN3 thymocytes. The proteostat dye specifically intercalates into the cross-beta spine of quaternary protein structures typically found in misfolded and aggregated proteins, which inhibits the dye’s rotation and leads to a strong fluorescence (Figure 5L). Sel1l CKO DN3 thymocytes displayed more protein aggregates compared with control thymocytes (Figure 5M,N). Collectively, these results corroborate that DN thymocytes lacking SEL1L accumulate misfolded/unfolded proteins leading to proteotoxic stress that then trigged the UPR and eventually apoptosis.

To determine whether upregulation of the UPR contributed to post-β-selected thymocyte apoptosis and the Sel1l CKO mouse phenotype, we generated Sel1l/Xbp1 double-knockout (Sel1^flox/flox.Xbp1^flox/flox.CD2-iCre), and Sel1l/Perk double-knockout (Sel1^flox/flox.Perk^flox/flox.CD2-iCre) mice. Deletion of Xbp1 in Sel1l CKO mice did not rescue thymocyte development (Figure 6—figure supplement 1A). In fact, Sel1l/Xbp1 double-knockout (DKO) mice showed more severe thymocytes development defects including a more than 95% loss in thymus cellularity and DP thymocyte numbers (Figure 6—figure supplement 1A). The DN3 and DN4 thymocytes from Sel1l/Xbp1 DKO exhibited much more Chop expression (Figure 6—figure supplement 1B). These data suggest that induction of the IRE1α/XBP1 pathway functions as a compensatory adaptative pathway to restrain Sel1l-deficiency induced ER stress.

In contrast to deletion of Xbp1, we found that deletion of Perk significantly rescued the Sel1l CKO mouse phenotype evident in the near complete restoration of thymus cellularity, DP and SP thymocyte cell numbers (Figure 6A,B). Perk deletion also restored peripheral T cells in spleen and lymph nodes compared to Sel1l CKO mice (Figure 6C–F). Consistent with the rescue, Perk deletion significantly reduced Sel1l-deficiency induced Chop induction (Figure 6G) and thymocyte apoptosis (Figure 6H,I) and restored normal cell cycle kinetics to Sel1l CKO DN4 thymocytes (Figure 6—figure supplement 1C). As Perk deficiency alone had no effect on T cell development (Figure 6A–I), these results indicate that activated PERK signaling contributed to the apoptosis of DN3/DN4 thymocytes that impaired β-selection in Sel1l CKO mice. Collectively, we conclude that SEL1L-ERAD promotes β-selected DN thymocyte differentiation by maintaining ER proteostasis and suppressing ER stress-induced cell death through the PERK pathway.

The mice were maintained in a pure C57BL/6 background and kept under specific-pathogen-free conditions in the transgenic mouse facility of the Baylor College of Medicine (22–24°C, 30–70% humidity with a 12 hr dark and 12 hr light cycle). Sel1l^flox/flox and Xbp1^flox/flox were described previously (Xu et al., 2020), Perk^flox/flox mice were purchased from Jackson Laboratory (Stock No. 023066). The floxed mice were crossed with either hCD2-iCre (The Jackson Laboratory, 008520) or Cd4-Cre (The Jackson Laboratory, 022071) mice to generate Sel1l^flox/flox; CD2-iCre, Sel1l^flox/flox; Cd4-Cre, Xbp1^flox/flox; CD2-iCre, or Perk^flox/flox; CD2-iCre mice. The Sel1l^flox/flox; Xbp1^flox/flox; CD2-iCre mice and Sel1l^flox/flox; Perk^flox/flox; CD2-iCre mice were generated by crossing Sel1l^flox/flox; CD2-iCre mice with Xbp1^flox/flox or Perk^flox/flox mice respectively. Atf6^flox/flox; Vav1-iCre mice were generated by crossing Atf6l^flox/flox (The Jackson Laboratory, 028253) with Vav1-iCre mice (The Jackson Laboratory, 008610). Sel1l CKO; OTII transgenic mice were generated by crossing Sel1l CKO mice with OTII transgenic mice (The Jackson Laboratory, 004194).

Six- to 8-week-old gender-matched mice were used for phenotype analysis and in vitro assay. For the bone marrow transplantation assays, female C57BL/6-Ly5.1 (CD45.1⁺) mice (Charles River, 564) were used at 8–12 weeks of age. All procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee or Case Western Reserve University Institutional Animal Care and Use Committee. The study is compliant with all of the relevant ethical regulations regarding animal research.

For the competitive bone marrow transplantation experiments, CD45.1⁺-recipient mice were lethally irradiated (10 Gy, delivered with two equal doses 4 hr apart) and injected retro-orbitally with 1x10⁶ whole bone marrow cells from Sel1l^flox/flox or Sel1l^flox/flox; CD2-iCre mice with an equal number of CD45.1⁺ competitor BM cells. For the BrdU incorporation assay, the mice were injected intraperitoneally with single dose of BrdU (BD, 559619; 50 mg/kg body weight) 2 hr before euthanization.

Single-cell suspensions from thymus, spleen and lymph nodes were obtained by passing the tissues through a 70 μm strainer. Bone marrow was removed from femurs and tibiae by flushing with PBS, 2% FBS. For bone marrow and spleen cell suspensions, red blood cells were removed by incubating with RBC Lysis Buffer (Biolegend, 420301). 1 × 10⁶ to 2 × 10⁶ cells were stained with antibodies at 4°C for 30 min in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA), followed by washing to remove the unbound antibodies.

Antibodies used for flow cytometry: The biotin-conjugated lineage markers for excluding non-DN cells are purchased from BioLegend: CD11b (M1/70, 101204), CD11c (N418, 117303), Ter119 (Ter119, 116204), Gr-1 (RB6-8C5, 108404), CD49b (DX5, 108904), and B220 (RA3-6B2, 103204). CD4 (BV650, RM4-5, 100546, BioLegend), CD8 (AF700, 53–6.7, 100730, BioLegend), CD25 (PE-Cy7, 3C7, 101915, BioLegend), CD44 (PE594, IM7, 103056, BioLegend), c-Kit (APC-Cy7, 2B8, 105838, Biolegend), γδTCR (PE, GL3, 118108, Biolegend), and CD27 (APC, LG.3A10, 124211, BioLegend) antibodies were used for analysis of ETP to SP cells in the thymus. CD4 (BV650, RM4-5, 100546, BioLegend), CD8 (AF700, 53–6.7, 100730, BioLegend), γδTCR (APC, GL3, 118115, BioLegend), B220 (PB, RA3-6B2, 103230, BioLegend), and NK1.1 (FITC, PK136, 108706, BioLegend) antibodies were used for the analysis of mature cells in the spleen and lymph nodes. Anti-CD45.1 (FITC, A20, 110706, BioLegend) and CD45.2 (APC, 104, 109814, BioLegend) antibodies were used for the analyses of donor chimerism in the bone marrow transplantation assay. All antibodies used in this study are listed in Appendix 1. Dead cells were excluded by 4,6-diamidino-2-phenylindole (DAPI) staining.

For cell sorting, DN thymocytes were purified by negative selection using a magnetic bead/column system (CD4 (L3T4) MicroBeads, 130-117-043; CD8a (Ly-2) MicroBeads, 130-117-044; LD Columns, 130-042-901; all purchased from Miltenyi Biotec) in accordance to the manufacturer’s instructions. The pre-enriched DN cells were further stained as described above to sort DN2, DN3, or DN4 cells. Dead cells were excluded by DAPI staining.

Flow cytometry data were collected using BD FACS Diva eight on a BD LSR II or BD Fortessa analyzer. The cell-sorting experiments were performed on a FACS Aria II cell sorter (BD). The acquired data were analyzed using the FlowJo 10 software.

For Annexin V staining, thymocytes were first stained with surface makers and then stained with Annexin V antibody for 30 min at room temperature in Annexin V binding buffer (TONBO, TNB-5000-L050). The cells were resuspended in Annexin V binding buffer with 1 μg/ml DAPI for analysis.

For cell cycle analysis, thymocytes were stained with surface markers followed by fixation and permeabilization with eBioscience Transcription Factor Staining Buffer Set (Life Technologies, 00-5523-00). The cells were then stained with anti-Ki-67 (FITC, 16A8, 652409, Biolegend) and DAPI for cell cycle analysis. For BrdU staining, total thymocytes were stained with surface markers to define each subset, followed by intracellular staining using the BrdU staining kit according to the manufacturer’s instructions (BD Biosciences, 559619).

OP9 bone marrow stromal cells expressing the Notch ligand DL-1 (OP9-DL1) were kindly provided by Dr. Juan Carlos Zúñiga-Pflücker (University of Toronto). The OP9-DL1 cells were cultured and maintained in αMEM medium, supplemented with 10% FBS (Gibco), penicillin (100 μg/mL), and streptomycin (100 U/mL) (Invitrogen). For thymocytes co-cultures, the sorted DN2 or DN3 cells were plated onto confluent OP9-DL1 monolayers (70–80% confluent) with addition of 5 ng/ml recombinant murine interleukin-7 (PeproTech) and 5 ng/ml Flt3L (PeproTech). Cells were harvested at different time points, filtered through 40 μm cell strainer, and stained with antibodies including ani-CD45 V450 (Tonbo, 75–0451 U100), anti-CD4 BV650 (Biolegend, 100546), anti-CD8a AF700 (Biolegend, 100730), anti-CD44 FITC (Biolegend, 103006), or anti-CD25 APC (Biolegend, 101910), followed by staining with Annexin V PE (Biolegend, 640908) and DAPI (4,6-diamidino-2-phenylindole, Invitrogen, D1306). EL4 cells (ATCC-TIB39) and HEK293T cells (ATCC CRL-3216) were cultured in DMEM with 10% FBS (Gibco), penicillin (100 μg/mL) and streptomycin (100 U/mL) (Invitrogen). No cell lines were listed in the database of cross-contaminated or misidentified cell lines by International Cell Line Authentication Committee (ICLAC). Cell lines from ATCC were authenticated by the STR profiling method and tested as mycoplasma contamination free by ATCC.

Thymus was fixed in fresh 4% paraformaldehyde for 24 hr and stored in 70% ethanol until paraffin embedding. Hematoxylin and eosin staining were performed on 5 μm–thick paraffin sections. For cleaved caspase-3 IHC, the anti-cleaved caspase-3 antibody (1:50, Cell Signaling Technology #9661) was used. Slides were incubated with Envision Labeled Polymer-HRP Anti-Rabbit (Dako, K4002) for 30 min. Sections were developed with DAB+ solution (Dako, K3468) and counterstained with Harris Hematoxylin. Imaging analysis was performed with ImageJ (FIJI) to automatically count the labeled cells in each region, the data were shown as number of positive cells per region.

Thymocytes were sorted directly into TRIzol LS reagent (Invitrogen, 10296010). Total RNA was extracted according to the manufacturer’s instructions. Total RNA was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368813). Quantitative real-time PCR was performed using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, A25778) on QuantStudio six real-time PCR system (Applied Biosystems). The primer sequences are listed in Appendix 1.

Western blot was performed as described previously (Xu et al., 2020). Approximately 5 × 10⁵ DN3 cells were sorted directly into 250 μl PBS containing 20% trichoracetic acid (TCA). The concentration of TCA was adjusted to 10% after sorting and cells were incubated on ice for 30 min before centrifugation at 13,000 rpm for 10 min at 4°C. Precipitates were washed twice with pure acetone (Fisher scientific, A18-4) and solubilized in 9 M urea, 2% Triton X-100, and 1% dithiothreitol (DTT) in 1 x LDS sample buffer (Invitorgen, NP0007). Samples were separated on NuPAGE 4–12% Bis-Tris protein gels (Invitrogen, NP0336BOX) and transferred to PVDF membrane (Millipore). The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. Blots were developed with the SuperSignal West Femto chemiluminescence kit (Thermo Scientific, 34096). Antibodies and reagents used are in Appendix 1. The original western blot images are in source data files.

DN3 thymocytes were directly sorted into TRIzol LS (ThermoFisher, cat. 10296028) and RNA was extracted following the standard protocol. The cDNA libraries were prepared using Truseq Stranded mRNA Kit (Illumina, California, USA # 20020594). Sequencing was performed on Illumina HiSeq 2000 (Illumina, California, USA), 150 bp paired end. Quality control was performed using FastQC. Raw reads were aligned to mouse genome GRC38 using STAR (2.5.2b) and counts for each protein-coding gene were obtained with HTSeq (2.7). DESeq2 package (1.26.0) in R (3.6.1) was used to perform analysis of differential gene expression. Upregulated genes (with adjusted p value < 0.05, log2 fold change > 0.59) were selected for Gene Ontology (GO) analysis using Enrichr (https://maayanlab.cloud/Enrichr/). We further performed Gene Set Enrichment Analysis (GSEA) using fgsea package (1.11.2) with padj-preranked gene lists and mouse gene set collection from Bader Lab. Heatmaps were generated with pheatmap (1.0.12).

For Figure 4C, cell culture plates were pre-coated with PBS or 5 μg/ml recombinant mouse DLL4 (BioLegend, #776706) overnight at 4°C before seeding EL4 cells. For Figure 4B,F–I,K and L, soluble DLL4 (5 μg/ml) was used. EL4 cells were incubated with soluble DLL4 for 24 hr before crosslinked with 1% formaldehyde for 10 min at room temperature. Reaction was quenched with 125 mM glycine. ChIP was performed as previously described (Zhao et al., 2018) with NOTCH1 NICD antibody (Abcam, ab27526), RBPJ antibody (Cell Signaling Technology, #5313), or normal rabbit IgG (Cell Signaling Technology, #2729). The sequences of all ChIP primers are listed in Appendix 1.

The firefly luciferase reporter for Sel1l or Hrd1 (Syvn1) promoter was constructed by cloning the genomic region into the MluI and XhoI sites or the MluI and HindIII sites in the pGL-3 basic vector (Promega), respectively. Mutations were made by overlap extension polymerase chain reaction as previously described (Bryksin and Matsumura, 2010). All constructs were verified by DNA sequencing. The sequences of all primers are listed in Appendix 1. HEK293T cells were transfected with Sel1l or Hrd1 promoter constructs, pRL-PGK (Promega) and 3xFlag-NICD1 (Addgene, #20183) or empty using Lipofectamine 3000 (Invitrogen, L3000015). Cell lysates were collected 48 hr after transfection, and luciferase activities were analyzed using the dual-luciferase reporter assay system (E1910, Promega). pRL-PGK, which expresses Renilla luciferase, was used as the internal control for adjustment of discrepancies in transfection and harvest efficiencies. EL4 cells were transfected with Sel1l or Hrd1 promoter constructs and pRL-PGK (Promega) using Lipofectamine 3000 (Invitrogen, L3000015). Cells were incubated with PBS or 5 μg/ml recombinant mouse DLL4 (BioLegend, #776706) for 24 hr before analysis.

A total of 5 × 10⁵ DN3 thymocytes were sorted from control (Ctrl, Sel1l^flox/flox) or Sel1l CKO (Sel1l^flox/flox; CD2-iCre) thymus and subjected to genomic DNA isolation using PureLink Genomic DNA Mini Kit (K1820-02) according to the manufacturer’s instructions. The amplification of eF-1 fragment was used as input control. The primers used for Vβ5-Jβ2, Vβ8- Jβ2, Vβ11-Jβ2, and eF1 are listed inAppendix 1. PCR products were resolved by 2% agarose gel electrophoresis.

Sorted DN3 thymocytes were washed with PBS, incubated with 1 μM ER-Tracker Green (Thermo Fisher, E34251) in PBS for 15 min at 37°C. The cells were then washed and resuspended in PBS, and analyzed by flow cytometry.

5x10⁶ thymocytes were stained for cell surface markers as described above. After surface markers staining, cells were washed twice with PBS. Tetraphenylethene maleimide (TMI; 2 mM in DMSO) was diluted in PBS to reach 50 μM final concentration and stained samples for 30 min at 37°C. Samples were washed once with PBS and analyzed by flow cytometry.

O-propargyl-puromycin (OPP; MedChem Source LLP) stock dissolved in 10% DMSO/PBS was diluted in PBS for intraperitoneal injection at 50 mg/kg. Mice were weighed, individually injected with OPP or vehicle, and euthanized 1 hr after injection. Thymuses were immediately harvested. Thymocytes were isolated and stained for surface antigens and viability dyes, which was followed by fixation and permeabilization according to the Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit (ThermoFisher Scientific). Briefly, after incubation cells were subsequently fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton-X100, then incubated with the AF647 reaction cocktail. Samples were acquired using a BD LSRFortessa and analyzed using FlowJo (Becton Dickinson) as per flow cytometry methods.

The PROTEOSTAT Aggresome Detection kit (Enzo Life Sciences, ENZ-51035–0025) was used to detect protein aggregates in freshly sorted DN3 thymocytes according to the manufacturer’s instructions. DN3 thymocytes were fixed, permeabilized and incubated with PROTEOSTAT dye (1:10,000 dilution) for 30 min at room temperature. Nuclei were counterstained with DAPI. Samples stained with DAPI only were used as negative controls. Images (16-bit greyscale TIFFs) were analyzed using CellProfiler v2.2. In brief, the DAPI channel images were first smoother with a median filter and nuclei identified with automatic thresholding and a fixed diameter. Nuclei touching the border of the image are eliminated. Touching nuclei are separated with a watershed algorithm. Then, cell boundaries were identified by watershed gradient based on the dye signal, using nuclei as a seed. Metrics were extracted from the cell, cytoplasm, and nuclear compartments.

TUNEL staining was performed on paraffin-embedded tissue sections using the In Situ Cell Death Detection Kit (catalog 11684795910, Roche) following the manufacturer’s instructions. Sections were counterstained with DAPI, and images were captured under fluorescence microscope. Tissue sections incubated with TUNEL reaction buffer without dTd enzyme served as negative controls. Tissue sections treated with DNase I served as positive controls. The quantification of TUNEL⁺ cells was performed with ImageJ (FIJI) to automatically count the labeled cells in each region. The data were presented as number of positive cells per region.

Data are expressed as the mean ± s.d. or mean ± s.e.m. as indicated in the figure legends; n is the number of independent biological replicates, unless specifically indicated otherwise in the figure legend. The respective n values are shown in the figure legends. The mice used for bone marrow transplantation were randomized and no blinding protocol was used. No statistical method was used to pre-determine the sample sizes. The results were quantified using GraphPad Prism 8. Student’s t test was utilized to compare the differences between two groups. One-way ANOVA with Tukey’s or Bonferroni’s multiple comparison test was used to compare the differences among three or more groups. Two-way ANOVA with Bonferroni’s post test was used to calculate the significance for in vitro DN2 differentiation measurement over time.

All protocols described in this study were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (protocol: AN-6813) or Case Western Reserve University Institutional Animal Care and Use Committee (protocol: 2017–0055).