The Ube2m-Rbx1 neddylation-Cullin-RING-Ligase proteins are essential for the maintenance of Regulatory T cell fitness

Wu, Di; Li, Haomin; Liu, Mingwei; Qin, Jun; Sun, Yi

doi:10.1038/s41467-022-30707-8

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Published: 31 May 2022

The Ube2m-Rbx1 neddylation-Cullin-RING-Ligase proteins are essential for the maintenance of Regulatory T cell fitness

Nature Communications volume 13, Article number: 3021 (2022) Cite this article

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Abstract

Neddylation-mediated activation of Cullin-RING E3 Ligases (CRLs) are necessary for the degradation of specific immune regulatory proteins. However, little is known about how these processes govern the function of regulatory T (Treg) cells. Here we show that mice with Treg cell-specific deletion of Rbx1, a dual E3 for both neddylation and ubiquitylation by CRLs, develop an early-onset fatal inflammatory disorder, characterized by disrupted Treg cell homeostasis and suppressive functions. Specifically, Rbx1 is essential for the maintenance of an effector Treg cell subpopulation, and regulates several inflammatory pathways. Similar but less severe phenotypes are observed in mice having Ube2m, a neddylation E2 conjugation enzyme, deleted in their Treg cells. Interestingly, Treg-specific deletion of Rbx2/Sag or Ube2f, components of a similar but distinct neddylation-CRL complex, yields no obvious phenotype. Thus, our work demonstrates that the Ube2m-Rbx1 axis is specifically required for intrinsic regulatory processes in Treg cells; and that Rbx1 might also play Ube2m-independent roles in maintaining the fitness of Treg cells, suggesting a layer of complexity in neddylation-dependent activation of CRLs.

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Introduction

CRLs are a family of multi-unit E3 ubiquitin ligases complex, consisting of 4 subunits: a scaffold cullin with 8 family members, an adaptor with many members, a substrate receptor with hundreds of members, and a RING component with two family members, RING-box 1 (Rbx1/Roc1) and RING-box 2 (Rbx2/Roc2/Sag)¹. The two members of RING family act as the catalytic subunit: Rbx1 couples with CRLs1–4, whereas RING-box 2 (Rbx2/Sag) couples mainly with CRL5². By promoting ubiquitylation of roughly 20% of cellular proteins doomed for proteasome degradation³, CRLs, as the largest family of E3 ubiquitin ligases, play an essential role in the regulation of apoptosis, cell cycle progression, DNA replication, DNA damage response and repair, gene transcription, genome integrity, signal transduction, stress responsiveness, and tumorigenesis^4,5.

Activity of CRLs requires neddylation on their cullin subunit, catalyzed by neddylation enzyme cascades, including E1 NEDD8 activation enzyme (NAE), a heterodimer of NAE1/APPBP1 and UBA3/NAEβ; E2 NEDD8 conjugating enzyme with two family members UBE2M and UBE2F, and several E3 NEDD8 ligases. Both Rbx1 and Rbx2 can also serve as co-E3 NEDD8 ligase⁶. It is well-established that UBE2M couples with RBX1 E3 to promote neddylation of cullins 1–4, whereas UBE2F couples with RBX2/SAG E3 for cullin-5 neddylation².

The total knockout studies from our laboratory have previously shown that Rbx1⁷ and Rbx2/Sag⁸, are functionally non-redundant and both required for mouse embryogenesis. Our recent study showed that RBX1 binds to CDC34/UBCH2C to promote ubiquitylation of large number of substrates via the K48 linkage, whereas RBX2 binds to UBE2C/UBE2S to promote ubiquitylation of different set of substrates via the K11 linkage for proteasome degradation^9,10. Recent studies showed that RBX1 also binds to UBCH5B/UBE2D2¹¹, UBCH5C/UBE2D3¹² E2s and ARIH1 E3¹³, whereas RBX2 binds to ARIH2 E3¹³, further demonstrating a new layer of difference between two family members.

A number of studies, using various cellular and mouse models, have shown that neddylaiton and CRLs regulate immune system via promoting ubiquitylation and degradation of specific immunological substrates. Examples include CRL1 regulation of NF-κB pathway¹⁴ and inflammatory cytokines¹⁵, CRL2^Vhl regulation of Treg cells via targeting Hif1α¹⁶, CRL3^SPOP regulation of PD-L1¹⁷, CRL5^Socs controlling of JAK/STAT pathway¹⁸, and Ube2m/Ubc12 regulation of efficient Th1 and Th2 differentiation of CD4⁺ T cells¹⁹.

While no conditional Rbx1 knockout mouse study was reported so far, our previous studies showed that T-cell specific Rbx2/Sag knockout (via Lck-Cre) significantly decreased T cell activation, proliferation, and T-effector cytokine release, although mice are viable²⁰; whereas Sag knockout in myeloid lineage (via LysM-Cre) increased serum levels of proinflammatory cytokines and enhanced mortality in response to LPS²¹. We have also shown the key role of neddylation in functional regulation dendritic cells (DC), given that neddylation inhibitor, MLN4924 suppressed the release of proinflammatory cytokines by DCs in response to various stimuli, and suppressed the ability of DCs to stimulate both murine and human allogeneic T cell responses²².

Regulatory T cells are specialized immunosuppressive CD4⁺-T lymphocytes, that play pivotal roles in maintaining immune homeostasis in vivo, illustrated by the early onset fatal auto-immune disorders upon depletion of Treg cells²³. Transcription factor Forkhead box P3 (Foxp3) is the master marker to distinguish the Treg cells from other CD4⁺-T cells^24,25,26, discovery of additional key regulators of Treg cells is required to gain a better understanding of fundamental biology of Treg cells. So far, there is no systematic study using mouse conditional knockout models to elucidate the physiological role of CRLs (via Rbx1/Rbx2) and neddylation (via Ube2m/Ube2f) in regulation of functions and survival of Treg cells for proper maintenance of homeostasis in immune system, even though both neddylation/CRLs and Treg cells play a fundamental role in immune modulation.

In this study, we investigate whether and how Rbx1-Rbx2/CRLs and Ube2m-Ube2f/neddylation regulate the homeostasis and function of Treg cells, and find that while mice with a Treg-specific deletion of Rbx2/Sag or Ube2f show no obvious phenotypes, mice with Rbx1 deletion in Treg cells develop an early-onset fetal inflammatory disorder and death at day ~25 after birth (~p25), with impaired suppressive functions and disrupted homeostasis of Treg cells, indicating Rbx1 as a prominent regulator of Treg cells. Moreover, mice with Ube2m deletion in Treg cells also suffers from inflammatory disorders, but to a much lesser severity with a 50% of death rate at ~150 days of age. The immune suppressive function of Treg cells deficient of either Rbx1 or Ube2m are severely or moderately compromised, respectively. Unbiased transcriptome comparison between Rbx1-deficient and Ube2m-deficient Treg cells reveals an overlapping but also unique changes in the signaling pathways controlling the inflammatory responses with the former showing greater alterations. Our study suggests that targeting CRL-neddylation could be an effective approach for the treatment of human diseases with over-activated Treg cells.

Results

Early-onset fatal inflammation in Foxp3 ^cre;Rbx1 ^fl/fl mice

To investigate the role of CRLs in the regulation of Treg cells in vivo, we generated conditional knockout mouse models with inactivation of Rbx1 and Rbx2/Sag individually in Treg cells, driven by Foxp3^YFP-cre (Foxp3^cre)^27,28. The compound mice were designated as Foxp3^cre;Rbx1^fl/fl or Foxp3^cre;Sag^fl/fl mice, respectively. Although Sag was expressed in wt Treg cells, Treg-specific depletion of Sag (Supplementary Fig. 1a) does not obviously impair Treg cell function and survival at the steady state (Supplementary Fig. 1b–d). Specifically, compared to Foxp3^cre controls (wild-type/wt), Foxp3^cre;Sag^fl/fl mice were viable and healthy without obvious alterations in activation markers of CD4⁺Foxp3⁻ T cells (conventional T cells, or Tcon cells) (Supplementary Fig. 1b), the Tcon cell proliferation rate (Supplementary Fig. 1c), and the Treg/CD4⁺ ratio (Supplementary Fig. 1d), suggesting that the role of Sag in Treg cells at the steady state is, if any, not obvious.

Strikingly, deletion of Rbx1 in Treg cells (Supplementary Fig. 2a) was early-onset fatal. Compared to wt controls, Foxp3^cre;Rbx1^fl/fl mice showed an altered appearance as early as day 13–15 after birth (p13–15), with collapsed ears, festered skin, and reduced body size (Fig. 1a). The mice continued to lose weight dramatically after p15 (Fig. 1b) with a 50% or 100% of death rate at p25 or p37, respectively (Fig. 1c). Autopsy examination revealed that Foxp3^cre;Rbx1^fl/fl mice had swollen lymph nodes and spleens (Fig. 1d, e), with lymphocyte infiltration into multiple organs, including the skin, lung, stomach, liver, kidney, and colon (Supplementary Fig. 2b). The Foxp3^cre;Rbx1^fl/fl mice also had a decreased CD4⁺/CD8⁺ T-cell ratio (Supplementary Fig. 3a, b) and a significantly increased proportion of effector/memory T cells (CD44^hiCD62L^lo) among populations of Tcon cells (Fig. 1f and Supplementary Fig. 3c). Beyond CD4⁺-T cells, multiple types of immune cells, including CD8⁺ cytotoxic T cells, macrophages, dendritic cells, B lymphocytes, were all dramatically activated in Foxp3^cre;Rbx1^fl/fl mice (Supplementary Fig. 4), indicating a typical phenotype of autoimmune disease. Such robust inflammation observed in Foxp3^cre;Rbx1^fl/fl mice is reminiscent of Treg-deficient mice²³ or mice with loss-of-function mutations in the Foxp3 gene²⁹, suggesting Rbx1 is absolutely essential for Treg cells in vivo.

**Fig. 1: *Rbx1* deletion in Treg cells leads to an early-onset fatal inflammatory disorder.**

The levels of Ifn-γ and Il-4 were remarkably increased in Tcon cells by flow cytometry detection of single cell suspension from lymph-nodes (Fig. 1g and Supplementary Fig. 3d). In addition, the serum concentrations of T_H1/T_H2 cytokines (Ifn-γ, Il-2 for T_H1; Il-4, Il-5, Il6, Il-9, Il-10, and Il-13 for T_H2) (Supplementary Fig. 5a, b) and antibodies (IgA and IgG2a for T_H1; IgE and IgG1 for T_H2) (Supplementary Fig. 6a, b) were all elevated and, indicating a robust activation of T_H1/T_H2-immune responses. For T_H17 reaction, flow cytometry of single cell suspension from lymph-nodes revealed a two-fold and statistically significant increase of Il-17A in Tcon cells (Fig. 1h and Supplementary Fig. 3e), and serum concentrations of T_H17 antibodies (IgG3, IgG2b) were also elevated, although not reaching the statistical difference (Supplementary Fig. 6c). While the serum concentrations of T_H17 cytokines (Tnf-α, and GM-Csf) did not change obviously, the Il-17A level was actually reduced (Supplementary Fig. 5c), despite a robust inflammatory storm was evident with significantly increased amount of various cytokines in Foxp3^cre;Rbx1^fl/fl mice (Supplementary Fig. 5d). Thus, while the T_H17 reaction was activated modestly, an excessive T_H1/T_H2-dominant immune polarization was clearly shown in Foxp3^cre;Rbx1^fl/fl mice.

Rbx1 is required for the immune suppressive function of Treg cells

To investigate whether the suppressive function of Rbx1-deficient Treg cells was impaired, we performed an in vivo suppression assay. The equal number of Treg cells from wt or Rbx1-deficient mice were injected into immune-deficient Rag1^−/− mice, along with naive conventional CD4⁺ T cells. If mice develop the colitis in 4 weeks, induced by co-injected naive conventional CD4⁺ T cells, it would indicate that Treg cells from Rbx1-deficient mice have lost suppression function, and vice versa. We found that while wt Treg cells prevent the colitis, as evidenced by a normal colon morphology, the Rbx1-deficient Treg cells failed to do so, and the mice developed very severe colitis, similar to the control mice without receiving Treg cell injection (Fig. 2a), demonstrating that the immune suppressive function of Treg cells was indeed impaired upon Rbx1-deletion.

**Fig. 2: Impaired suppressive function of Rbx1-deficient Treg cells.**

To exclude the possibility that Rbx1-deficient Treg cells might die in the 4-week period during the in vivo suppression assay, we also used an alternative strategy to investigate the impairment of the immune suppressive function of Rbx1-deficient Treg cells at early age. Compared to the wt control mice, Foxp3^cre;Rbx1^fl/fl mice at the p8 stage have similar levels of Treg/CD4⁺ ratio (Fig. 2b), similar numbers of Treg cells, as well as total numbers of lymphocytes (Fig. 2c, d). However, these Treg Rbx1-deficient infant mice still showed an over-activated immune system with a ~3–4-fold increase in proportion and cell number of effector/memory (CD44^hiCD62L^lo) T cells (Fig. 2e).

Finally, we performed the wt Treg infusion experiment and found that intraperitoneal injection of wt Treg cells to the infant Foxp3^cre;Rbx1^fl/fl mice cannot rescue the fatal inflammation (Fig. 2f, g). Specifically, compared to wt control, Foxp3^cre;Rbx1^fl/fl mice (at the p8 stage) transplanted with wt Treg cells still developed immune over-activation (harvested at the p20 stage), revealed by elevated proportion of effector/memory T cells (CD44^hiCD62L^lo) among populations of Tcon cells (Fig. 2f), and increased proliferation rate of Tcon cells (Fig. 2g). The possible explanation is that despite Treg cells was reported to inhibit established inflammation in 5 weeks³⁰, the infused wt Treg cells did not have enough time window for functional rescue after inflammation had already started in the recipient infant Foxp3^cre;Rbx1^fl/fl mice. Taken together, all these three assays demonstrated that Rbx1 is essential and required for immune suppression function of Treg cells.

In moribund Foxp3^cre;Rbx1^fl/fl mice at p19–23 stage, Treg cellular compartment was reduced dramatically, as evidenced by reduced Treg cell ratios (Supplementary Fig. 7a) and actual numbers (Supplementary Fig. 7b), with reduced proliferation and increased apoptosis, revealed by reduced Ki67 expression (Supplementary Fig. 7c) and increased Caspase-3 activity (Supplementary Fig. 7d). Thus, the fatal inflammatory disorders in Foxp3^cre;Rbx1^fl/fl mice are attributable to both impaired suppressive function and decreased cellular compartment.

Rbx1 is essential for the conversion of quiescent to effector subpopulations of Treg cells

Single-cell RNA sequencing (scRNA-seq) was utilized to unbiasedly dissect the cellular heterogeneity of wt vs. Rbx1-deficient CD4⁺YFP⁺ Treg cells from the peripheral lymph nodes of inflammation-free female Foxp3^cre/wt and Foxp3^cre/wt;Rbx1^fl/fl mice (Note that the Foxp3 gene is localized in the X-chromosome, and the female mice used here with two genotypes are chimeric, Supplementary Fig. 8). As revealed in the two-dimensional Uniform Manifold Approximation and Projection (UMAP) of the single-cell transcriptomics data, both wt and Rbx1-deficient Treg cells could be divided into several subpopulations (Fig. 3a and Supplementary Fig. 9). Rbx1-depletion increased the subpopulation in Cluster 0, but remarkably decreased the subpopulations on Clusters 3 and 4 without obviously affecting other subpopulations (Fig. 3a, b).

**Fig. 3: Single-cell transcriptomics of wt and *Rbx1*-deficient Treg cells.**

The top 10 genes and top 10 pathways in each of 14 clusters in Treg cells were shown (Supplementary Figs. 10, 12). Among them, the cells in Cluster 0 expressed high levels of genes characteristic of naive Treg cells, including Sell (Cd62L)³¹ and Bcl2³²; while Treg cell functional molecules, such as Ctla4³³, Icos³⁴, and Cd44³⁵, were highly expressed in Cluster 3 and 4 (Fig.3c). To confirm this finding, we performed the FACS-based profiling and found a ~3–4-fold reduction of CD44^hiCD62L^lo cells in Treg population from peripheral lymph nodes in Foxp3^cre;Rbx1^fl/fl mice at age of ~20-day (Fig. 3d and Supplementary Fig. 11a). A similar reduction was also observed in infant Foxp3^cre;Rbx1^fl/fl mice at age of 8-day (Fig. 3e and Supplementary Fig. 11b), indicating that the reduction in Cluster 3/4 subpopulations upon Treg Rbx1-deletion is an early event.

The KEGG pathway enrichment analysis revealed that, compared to Cluster 0, T-cell receptor³⁶ and other Treg-related pathways were activated in Clusters 3 and 4 (Fig. 3f and Supplementary Fig. 12). Thus, Cluster 0 is a quiescent subpopulation, designated as the center population, whereas Clusters 3 and 4 are active Treg cell subpopulations, designated as effector populations³⁷. The fact that the loss of Rbx1 leads to the failure in the conversion of quiescent subpopulation to effector subpopulation supports the notion that Rbx1 is essential for such a conversion in the Treg cells.

Disordered functional and regulatory network in Rbx1-deficent Treg cells

Transcriptome analysis of Rbx1-deficient Treg cells from inflammation-free female Foxp3^cre/wt;Rbx1^fl/fl mice (Supplementary Fig. 8) revealed significant down-regulation of functional genes, including Il10²⁷, Tgfb³⁸, and Cst7³⁹ (suppressive cytokines); Il2r⁴⁰ and Entpd1^41,42 (regulators of immune cell metabolism); Ctla4³³, Icos³⁴, Nrp1⁴³, and Lag3⁴⁴ (inhibitor via dendritic cells); and Fas⁴⁵ and Lgals1⁴⁶ (apoptosis inducer). Impairment of migration-associated surface molecules was also observed in Rbx1-deficient Treg cells, including downregulation of Cd44, Cxcr3, Cxcr5, and Cxcr6³⁵ and upregulation of Sell³¹ (Fig. 4a).

**Fig. 4: Rbx1-dependent transcriptome and proteome in Treg cells.**

The KEGG pathway enrichment analysis revealed that Rbx1 depletion caused downregulation of transcriptome involved in many signaling pathways, including T-cell receptor, Th17 cell differentiation, chemokine and cytokine–cytokine receptor interaction, NOD-like receptor, TNF, FoxO, Toll-like receptor, Hippo, TGFβ, NF-κB, PI3K-AKT, and purine metabolism among few other virus associated pathways (Fig. 4b and Supplementary Fig. 13). The results suggest that loss of Rbx1 results in changes in the expression of numerous key genes regulating the processes of inflammation, immunological responsiveness, proliferation/survival, and metabolisms, which are subjected to further experimental validation.

Considering Rbx1 is an E3 ubiquitin ligase that directly controls the abundance of substrate proteins, we utilized the mass spectrometry-based proteomics to profile the difference in protein levels between wt and Rbx1-deficient CD4⁺YFP⁺ Treg cells (three independent sets of combined samples) from inflammation-free female Foxp3^cre/wt and Foxp3^cre/wt;Rbx1^fl/fl mice (Supplementary Fig. 8). Among a total of 3236 proteins detected in any one of 6 paired samples, we identified 43 increased and 78 decreased proteins with a three-fold cut-off, and the numbers increased to 70 and 195, respectively, with a two-fold cut-off in Rbx1-deficient CD4⁺YFP⁺ Treg cells, as compared to the wt control. The KEGG pathway enrichment analysis of two-fold cut-off revealed that Rbx1 depletion caused an obvious change of many pathways (Supplementary Fig. 14), including accumulation of few proteins associated with the splicesome or complement and coagulation cascade, but significant reduction of proteins involving ER processing, and of proteins controlling the various metabolic pathways, including TCA cycle, carbon and purine metabolisms, along with other metabolic pathways (Fig. 4c, d). We marked (with arrows) the genes with the consistent changes at both mRNA and protein levels, and these observed changes at the protein levels are likely due to altered gene transcription (Fig. 4c, d), which is again subjected to further experimental validation.

Specific spectrum of Rbx1 substrates in Treg cells

To define candidates as possible direct substrates of Rbx1 ubiquitin E3 ligase in Treg cells, the proteomic and transcriptomic data were compared directly. We reasoned that the substrate candidates should be those with the increase at the levels of protein, but not of mRNA, in Rbx1-deficient Treg cells. A total of 57 such candidates were identified (Fig. 5a). While Acly⁴⁷, Bcl2l11/Bim⁴⁸, Ets1⁴⁹, Nfkbib/IκB-β⁵⁰, and Zhx2⁵¹ were previously reported as Rbx1 substrates, suggesting the reliability of our methodology, several others in the list are likely the novel substrates for future validation.

**Fig. 5: Comparison of the levels of proteins vs. mRNAs of indicated genes in CD4⁺YFP⁺ Treg cells from *Foxp3*^cre/wt and *Foxp3*^cre/wt;*Rbx1*^fl/fl mice (8–10 weeks old).**

Interestingly, many common substrates of Rbx1/CRLs1-4^1,6 were not detected in Rbx1 deficient Treg cells, and few detected ones, like Cdkn1b/p27⁷ and Foxo1⁵², were not accumulated (Fig. 5b). Thus, it appears that only a small subset of Rbx1 substrates are selectively expressed, and specifically subjected to Rbx1 modulation in Treg cells to maintain their proper functions.

The involvement of Bim in the phenotypes of Foxp3 ^cre;Rbx1 ^fl/fl mice

In order to validate these putative Rbx1 substrates in Treg cells, we searched all 57 proteins for antibodies commercially available for FACS-based quantification, given the difficulty in harvesting sufficient Treg cells for traditional Western blotting analysis. Unfortunately, only available antibody is for Bim. We, therefore, performed the FACS analysis, as described⁵³, to quantify the levels of Bim, and confirmed that Bim is indeed accumulated up to three-fold in Rbx1-depleted Treg cells (Fig. 6a, b). It is noting worthy that our single cell RNA sequencing data revealed that Bim mRNA is widely expressed in all subpopulations of Treg cells (Supplementary Fig. 15a).

**Fig. 6: Involvement of Bim in the phenotypes of *Foxp3*^cre;*Rbx1*^fl/fl mice.**

Given Bim is a pro-apoptotic protein⁵⁴, and also involved in Treg cells regulation⁵⁵, Bim accumulation could mediate at least in part some phenotypes of Rbx1-deficient Treg cells. We tested this working hypothesis via a Bim rescue experiment by generating and characterizing conditional knockout mice with inactivation of both Rbx1 and Bim in Treg cells driven by Foxp3^YFP-cre (Foxp3^cre)^27,28 (designated as Foxp3^cre;Rbx1^fl/fl;Bim^fl/fl mice), which have more than 10-fold reduction of Bim levels in Treg cells (Supplementary Fig. 15b). The Foxp3^cre;Rbx1^fl/fl;Bim^fl/fl mice also developed an early-onset fetal inflammatory disorder with very short lifespan, similar to the Foxp3^cre;Rbx1^fl/fl mice (Fig. 6c, d), along with decreased CD4⁺/CD8⁺ T-cell ratio (Supplementary Fig. 15c) and significantly increased proportion of effector/memory T cells (CD44^hiCD62L^lo) among populations of Tcon cells (Fig. 6e and Supplementary Fig. 15d). Notably, the above inflammation markers, however, were milder in double cKO Foxp3^cre;Rbx1^fl/fl;Bim^fl/fl mice than those in Foxp3^cre;Rbx1^fl/fl mice, with the statistically significant p values (Fig. 6e, f), suggesting the inflammation and immune over-activation induced by Rbx1-deficiency in Treg cells were moderately attenuated by Bim-deletion. Moreover, Treg ratio among CD4⁺-T cells in Foxp3^cre;Rbx1^fl/fl;Bim^fl/fl mice remained significantly reduced, as compared to wt control (Fig. 6g and Supplementary Fig. 15e), despite slightly higher than that in Foxp3^cre;Rbx1^fl/fl mice (Fig. 6h). However, the absolute numbers of Treg cells in Foxp3^cre;Rbx1^fl/fl;Bim^fl/fl mice were comparable to the wt control mice (Supplementary Fig. 15f), while it was significantly reduced in Foxp3^cre;Rbx1^fl/fl mice (Supplementary Fig. 7b). Notably, Treg cells with double deletion of Rbx1 and Bim still suffer from elevated apoptosis (Supplementary Fig. 15g), in a level similar to Rbx1-deficient Treg cells (Supplementary Fig. 7d). Taken together, Bim deletion in Treg cells attenuated the severe inflammation phenotype, although it failed to rescue the survival of Foxp3^cre;Rbx1^fl/fl mice, indicating Bim is partially involved in the function of Rbx1 in Treg cells.

Later-onset fatal inflammation in Foxp3 ^cre;Ube2m ^fl/fl mice with impaired suppressive function of Treg cells

It has been well-established that the E3 ligase activity of the CRLs requires neddylation of the scaffold protein Cullin^56,57. Specifically, neddylation E2 Ube2m couples with Rbx1 (neddylation co-E3) selectively promotes neddylation of Cullins 1–4, which are complexed with Rbx1 to constitute active CRLs 1–4 ubiquitin E3; whereas neddylation E2 Ube2f couples with Sag/Rbx2 E3 to promote neddylation of Cullin 5, which is then complex with Sag/Rbx2 to constitute active CRL5². We, therefore, asked whether the pivotal role of Rbx1 in Treg cells is dependent on Ube2m, whereas Ube2f is non-essential for Treg cells, given the non-essentiality of Sag/Rbx2 (Supplementary Fig. 1). Indeed, despite Ube2f is expressed in wt Treg cells (Supplementary Fig. 16a), Foxp3^cre;Ube2f ^fl/fl mice with Ube2f deletion in Treg cells are viable and develop normally without any abnormality, as evidenced by a normal level of an activation marker of CD4⁺Foxp3⁻ T cells (Supplementary Fig. 16b), a normal rate of Tcon cell proliferation (Supplementary Fig. 16c), and a normal Treg/CD4⁺ ratio (Supplementary Fig. 16d), indicating that the Ube2f-Rbx2-CRL5 axis is not essential for the functionality and survival of Treg cells at the steady status.

On the other hand, like Foxp3^cre;Rbx1^fl/fl mice, Foxp3^cre;Ube2m^fl/fl mice with Ube2m deletion in Treg cells (Supplementary Fig. 17a), displayed abnormal appearances such as collapsed ears and festered skin, but at the much later stage (Fig. 7a). The growth of mice was remarkably retarded (Fig. 7b), and about 50% of the mice dies at age of 150 days and a 100% death rate within one year (Fig. 7c). The swollen spleens and lymph nodes were apparent with increased cellularity (Fig. 7d and Supplementary Fig. 17b) with moderate lymphocyte infiltration into multiple organs, including skin, lung, stomach, liver, kidney, and colon (Supplementary Fig. 17c). Furthermore, the Foxp3^cre;Ube2m^fl/fl mice also showed obvious T_H1/T_H2-polarized immune over-activation of multiple immune cells (Fig. 7e–g and Supplementary Figs. 18–21), but in a much lower degree of inflammation than that in Foxp3^cre;Rbx1^fl/fl mice (Fig. 1 and Supplementary Figs. 2–6).

**Fig. 7: Fetal, but relative weaker, inflammatory disorder in *Foxp3*^cre;*Ube2m*^fl/fl mice.**

Interestingly, unlike Foxp3^cre;Rbx1^fl/fl mice, Foxp3^cre;Ube2m^fl/fl mice had increased Treg/CD4⁺-T ratio and increased number of Treg cells (Fig. 8a) in lymph nodes, without obvious alterations in the levels of Ki67 and active Caspase-3 in Treg cells (Fig. 8b, c). It has been shown that in response to inflammation insult, the immuno-suppressive Treg cells usually proliferate to inhibit the inflammation and restore the immune homeostasis of the body^58,59. The ratio (Treg/CD4⁺) and number of Treg cells are, therefore, elevated in vivo under inflammation, if the proliferation and survival of Treg cells are not inhibited, which is the case for Ube2m-deficient Treg cells, but not for Rbx1-deficient Treg cells. Nevertheless, the fact that Foxp3^cre;Ube2m^fl/fl mice still developed over-reactive immune response (Fig. 7) strongly suggested the impairment of suppressive function of Ube2m-deficient Treg cells. Indeed, the in vivo suppression assay using immune-deficient Rag1^−/− mice clearly showed that very severe colitis, induced by injection of naive CD4⁺-T cells, cannot be inhibited by Ube2m-deficient Treg cells (Fig. 8d). However, unlike that of Foxp3^cre;Rbx1^fl/fl mice, the inflammatory immune phenotype of Foxp3^cre;Ube2m^fl/fl mice can be largely rescued by intraperitoneal injection of wt Treg cells, as evidenced by a largely normal proportion of both effector/memory T cells (CD44^hiCD62L^lo) among populations of Tcon cells (Fig. 8e) and proliferation rate of Tcon cells (Fig. 8f), suggesting that impaired immune suppressive function in Foxp3^cre;Ube2m^fl/fl mice are relatively minor, as compared to Foxp3^cre;Rbx1^fl/fl mice and can be rescued by wt Treg cells in this infusion experiment.

**Fig. 8: Impaired suppressive function of Ube2m-deficient Treg cells.**

Comparison of signaling pathways regulated by Rbx1 and Ube2m in Treg cells

The phenotypes between Foxp3^cre;Rbx1^fl/fl and Foxp3^cre;Ube2m^fl/fl mice clearly demonstrate the similarities and differences between Rbx1 and Ube2m in the maintenance of the functionality of Treg cells. To dissect them at the molecular levels, we performed the genome-wide transcriptome analyses of CD4⁺YFP⁺ Treg cells derived from female Foxp3^cre/wt;Ube2m^fl/fl and Foxp3^cre/wt;Rbx1^fl/fl mice (Supplementary Fig. 8). The results showed the similar changes in the Treg functional genes (Supplementary Fig. 22), and multiple signaling pathways involved in inflammation responses, but generally to a lesser extent in Ube2m-deficient Treg cells (Supplementary Fig. 23). An unbiased cluster analysis of the transcriptomes between Rbx1-deficient and Ube2m-deficient Treg cells revealed a good overall correction, but two sets of data did have unique differences in several groups of genes and pathways, particularly seen in Rbx1-deficient Treg cells (Fig. 9a and Supplementary Figs. 24, 25), demonstrating that CRLs and neddylation did have common as well as unique mechanisms in the regulation of Treg cell functions.

**Fig. 9: Rbx1 acts via Ube2m-dependent and -independent mechanisms in Treg cells.**

Taken together, both the phenotypic comparison and unbiased cluster analyses of the transcriptome support the notion that Rbx1 plays Ube2m-dependent and -independent roles in Treg cells (Fig. 9b).

Discussion

In this study, we systematically investigated the role of CRLs and neddylation in the maintenance of fitness of Treg cells, using four Treg selective KO mouse models. While the Ube2f/Sag axis plays a minimal, if any, role, the Ube2m/Rbx1 axis is indeed essential for the homeostasis and survival of Treg cells, although all four genes are widely expressed in multiple mouse tissues⁶⁰ and in all subpopulations in Treg cells (Supplementary Fig. 26). The finding that mice of Rbx1 Treg deletion suffer from much more severe autoimmune disorders with much earlier fatality than those of Ube2m Treg deletion is unexpected, since Ube2m is an upstream E2, whereas Rbx1 is a downstream co-E3 in the neddylation cascade. Thus, our in vivo phenotypical study via targeting Treg cells clearly demonstrated that Rbx1/CRLs1–4 have additional functions beyond neddylation activation (Fig. 9b), as generally accepted, which was mainly based upon the in vitro biochemical and cell biology studies². Furthermore, severe autoimmune phenotype of Foxp3^cre;Rbx1^fl/fl mice is reminiscent of mice devoid of Treg cells²³ or deficient in Foxp3²⁹. Very few genes, upon Treg deletion in mice, have shown a severe autoimmune phenotype, similar to that of the Treg-deficiency, including Foxo1⁵⁸, Raptor⁵⁹, Lkb1⁵³, Uqcrsf1⁶¹, and Pggt1b⁶². Thus, Rbx1, encoding a dual E3s for ubiquitylation and neddylation, is a new addition to this short list as one of the most critical genes, absolutely required for the functionality of Treg cells.

Several lines of evidence suggest that severe autoimmune/inflammatory phenotype of Foxp3^cre;Rbx1^fl/fl mice is not the simple consequence of reduced proliferation and/or enhanced apoptosis, rather it is also attributable to the impaired immune suppression of Rbx1-deficent Treg cells. First, in an in vivo suppression assay, Rbx1-deficient Treg cells failed to inhibit the colitis induced by naive CD4⁺ T cells in immune-deficient Rag1^−/− mice (Fig. 2a); Second, the 8-day-old infant Foxp3^cre;Rbx1^fl/fl mice with normal ratio of Treg/CD4⁺-T cells and normal number of Treg cells already showed the immune activation (Fig. 2b–e); third, infusion of wt Treg cells into Foxp3^cre;Rbx1^fl/fl mice failed to rescue inflammation phenotype within the testing time window (Fig. 2f, g); and forth, simultaneous deletion of Bim and Rbx1 failed to extend mouse life-span and rescued the severe inflammation phenotypes (Fig. 6).

So far, the only reported study of CRL regulation of Treg cells was the Treg deletion of Vhl, a substrate receptor of CRL2 for targeted degradation of Hif1α⁶³. In that study, the authors showed that mice with Vhl-deficient Treg cells also developed severe inflammatory disorders with a 50% of death rate at the age of 8 weeks¹⁶. Mechanistically, the production by Vhl-deficient Treg cells of a large amount of Ifn-γ due to transactivation by accumulated Hif1α is responsible for immune-disorder phenotype, which was completely rescued by simultaneous Ifn-γ deficiency in Treg cells¹⁶. In contract, the production of Ifn-γ by Rbx1-deficient Treg cells is comparable with that of wt controls (Supplementary Fig. 27), although other T cells (non-Treg cells) produced more Ifn-γ (Fig. 1g and Supplementary Fig. 3d), indicating a different mechanism of action between Vhl and Rbx1 in functional regulation of Treg cells.

Our Microwell-based scRNA-seq revealed unbiasedly that Treg cells were divided into several subpopulations, and the quiescent population was significantly increased, whereas the effector subpopulations were significantly reduced upon Rbx1 depletion (Fig. 3), highly suggesting a failure in the conversion of the quiescent subpopulations into effector ones, rather than the death of these effector cells. Mechanistically, although we identified several altered signaling pathways (Fig. 3f), how exactly Rbx1 depletion failed to trigger such a conversion is currently unknown. Given the major role of Rbx1 as a dual E3 in both neddylation and ubiquitylation, it is very likely that Rbx1 depletion would result in the abnormal levels or activities of transcription factors and/or signaling molecules directly (via substrate accumulation or neddylation modification) or indirectly (altered transcriptions due to direct effect). Future studies are geared to elucidate the underlying mechanism. On the other hand, the fact that Rbx1-deficiency has no effect on other subpopulations of Treg cells provides an ideal opportunity of Rbx1 targeting for precise manipulation of Treg cell subpopulations.

It is worth noting that droplet-based and plate-based strategies were previously used for the scRNA-seq transcriptomics analysis of Treg cells³⁷. The subpopulations of Treg cells revealed by our study were largely in agreement with the previous report³⁷ with exception that the center and effector populations were combined as a single population in that report³⁷. The other minor differences are likely derived from different platforms. Nevertheless, a direct comparison between wt and Rbx1-null Treg cells indeed provided informative knowledge as to how Rbx1 regulates Treg subpopulations. Furthermore, this study is the first to provide detailed informative heat-map of top 10 characteristic genes in each of 14 clusters via scRNA sequencing, along with associated pathways (Supplementary Figs. 10, 12).

Both proteomics and transcriptome approaches were employed to analyze the effect of Rbx1-deficiency on Treg cells at the molecular levels. We noticed that less than half of protein changes seen in the proteomic analysis matched with the mRNA changes by the transcriptomic analysis (Fig. 4c, d). While this is slightly lower than the general correlation of 50% between proteome and transcriptome⁶⁴, the correlation between proteome and transcriptome is known to be distinct in different tissues or cell types⁶⁵, including in human Treg cells, as reported recently⁶⁶, likely due to the low abundance of Treg population in vivo with limited detection in our study of only ~3200 proteins in any given 6 samples by Mass Spectrum analysis. The comparison of proteome and transcriptome of Treg cells should provide some cues for future investigation of detailed mechanisms of Treg cell regulation by CRLs and neddylation.

The combination of proteomic and transcriptomic data revealed several Treg-specific Rbx1 degradation candidates, including Acly⁴⁷, Bcl2l11/Bim⁴⁸, Ets1⁴⁹, Nfkbib/IκB-β⁵⁰, and Zhx2⁵¹. Among the putative substrates of Rbx1 in Treg cells, Nfkbib/IκB-β is the inhibitor of NF-κB pathway, Fto is the demethylation enzyme of m⁶A mRNA methylation, Ets1 is a transcription repressor, whereas Bcl2l11/Bim is a pro-apoptotic protein. The NF-κB pathway^28,67, m⁶A mRNA methylation⁶⁸, and Bcl2l11/Bim⁵⁵ are all known to regulate Treg cell. Our Bim rescue experiment showed a partially role of Bim in Treg Rbx1-deficient phenotypes (Fig. 6), suggesting the severe inflammation phenotype is likely caused by the combination of multiple substrates. Future studies should be directed to characterize the substrates, particularly those involved in the regulation of global gene expressions (such as Fto and Ets1) in the functional regulation of Treg cells upon Rbx1 deletion.

While Treg Ube2m-deficiency also causes obvious inflammation disorders (Fig. 7), the phenotype severity is much lesser than that seen in Treg Rbx1-deficient mice (Fig. 1). Mechanistically, although both Rbx1 and Ube2m controls the expression of genes regulating several inflammation-related pathways (Supplementary Fig. 23), the downregulation of Egr2⁶⁹, Ccr8⁷⁰ and Tigit⁷¹ is rather unique to Rbx1-deficient Treg cells (Fig. 9a and Supplementary Fig. 24). Altered expression of these genes, which has been previously shown to regulate Treg cell function, would likely be responsible for the more severe phenotype seen in Foxp3^cre;Rbx1^fl/fl mice.

In summary, we used in this study three comprehensive approaches, namely single cell sequencing, whole transcriptome profiling, and mass-spect based proteomics, in an attempt to define the mechanism of Rbx1 action in Treg cells. Given the broad role of the neddylation-CRLs in fundamental biological processes, our study yielded a wealth of information on many altered signaling pathways and individual gene/protein upon Rbx1 Treg deletion. We reason that this is the first overall phenotypical study of elucidating the role of neddylation enzymes, particularly Rbx1/Ube2m, in Treg cells, which sets the foundation for future detailed studies to pinpoint special role of each interesting pathways and individual genes. Given that inactivation of either neddylation E2/Ube2m, or E3/Rbx1 caused suppression of Treg function, MLN4924, a small molecule inhibitor of neddylation, currently in several Phase II clinical trials for anticancer application^3,6 may have a new application in the treatment of diseases associated with over-activation of Treg cells, which is certainly worth investigating experimentally.

Methods

Mice

The Foxp3^YFP-cre mice (Jax No. 016959) are gifts from Prof. Xiaoming Feng (Peking Union Medical College, China).

The Rbx1^fl/fl mice were generated on the C57BL/6 background via CRISPR/Cas9 technology with exons 2–4 floxed, performed by GemPharmatech Co., Ltd. (Nanjing, China). Briefly, the sgRNA (5′-A CCACTAAGTGGATAATCAC-3′ and 5′-GGAGTCAGAAATGATTGGTC-3′) direct Cas9 endonuclease cleavage in intron1–2 and intron 4–5 to insert LoxP sites by homologous recombination. The PCR genotyping primers 5′-GATTCTACTTTGCTTGCAGTGCTC-3′ and 5′-TCTGCATAAGCACGGGCTCTC-3′ generated DNA fragment of 266 and 196 bp bands, respectively, for mutant and wild type.

The Ube2m^fl/fl mice were generated on the C57BL/6 background, performed by GemPharmatech Co., Ltd. (Nanjing, China). The CRISPR/Cas9 technology was used to flox exons 2–4 with exon 5 frame-shifted in Ube2m genome. Given exon 1 only encodes 36 amino acids, there is no possibility that a truncated functional protein is still being made. Briefly, the sgRNA (5′-TCAATTTAAGCTACCATAC-3′ and 5′-AGTGAAGATACGGGACA-3′) direct Cas9 endonuclease cleavage in intron1–2 and intron 4–5 to insert LoxP sites by homologous recombination. The PCR genotyping primers 5′-CAAGACCTGCCTTCCAGGTATC-3′ and 5′-CCCTGCTAATACTGAACAGG-3′ generated DNA fragment of 317 and 223 bp for mutant and wild-type, respectively.

The Ube2f^fl/fl mice were generated from an ES clone (HEPD0820_4_G09), purchased from EuMMCR (European Mouse Mutant Cell Repository) (MGI:1915171) with exon 5 floxed. For LoxP sites flanked allele, the PCR genotyping primers 5′-GCGAGCTCAGACCATAACTTCG-3′ and 5′-CCAGGGTGGAAAATTTCAGTTT-3′ generated DNA fragment of 320 bp; and for wild type allele, the genotyping primers 5′-CCCTGGAATTTCGGTATTATA-3′ and 5′-CCAGGGTGGAAAATTTCAGTTT-3′ generated DNA fragment of 320 bp, respectively.

The Sag^fl/fl mice were generated and characterized as described⁷².

The Bim^fl/fl mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China) (T007185).

The Rag1^−/− mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China) (T004753).

All mice were maintained in specific pathogen-free (SPF) conditions with experimental/control groups co-housed, and experiments were conducted with gender-matched experimental/control mice. Mice were euthanized via cervical dislocation or carbon dioxide. Permission was granted to perform animal experiments by the Animal Ethics Committee of Zhejiang University; animal care was provided in accordance with the principles and procedures by the regulatory standards at Zhejiang University Laboratory Animal Center. To request the mouse strains generated in this project, please contact Dr. Yi Sun, e-mail: yisun@zju.edu.cn.

Flow cytometry

Peripheral lymph nodes and/or spleens were harvested from mice with indicated genotypes, and grinded into single cells, subjected to followed experiments. For analysis of surface markers, cells were washed in PBS containing 2% (wt/vol) FBS, and stained with indicated Abs (dilution 1: 200); intracellular markers were strained with Transcription Factor Buffer Set (BD Pharmingen, 562574) according to the manufacturer’s instructions by indicated Abs (dilution 1: 50). For intracellular cytokine staining, cells were stimulated for 5 h with leukocyte activation cocktail of GolgiPlug (BD Pharmingen, 550583). Antibodies were all from eBioscience, unless otherwise noted: anti-CD4 (GK1.5), anti-CD8α (53-6.7, BD Pharmingen), anti-CD44 (IM7, BD Pharmingen), anti-CD62L (MEL-14), anti-Foxp3 (FJK-16s), anti-Ki67 (SolA15), anti-CD45 (30-F11), anti-F4/80 (BM8), anti-CD11b (M1/70), anti-CD11c (N418), anti-MHCII (M5/114.15.2), anti-CD80 (16-10A1), anti-CD86 (GL1, BD Pharmingen), anti-B220 (RA3-6B2), anti-CD19 (1D3, BD Pharmingen), anti-GL7 (GL7, BD Pharmingen), anti-Fas (15A7), anti-active caspase 3 (C92-605, BD Pharmingen), anti-Ifn-γ (XMG1.2), anti-Il4 (11B11), anti-Il17A (eBio17B7), and anti-Bim (C34C5, Cell Signal Technology). Flow cytometry data were acquired on CytoFLEX LX (Beckman) and analyzed using Flowjo software (Tree Star).

Cytokines assay

The serum was drawn from mice of indicated genotypes, diluted four-fold by PBS, and then subjected to cytokine/chemokine profiling using Mouse Cytokine Grp I (Bio-Plex Pro, M60009RDPD), measured on Luminex 200 system.

Antibodies assay

The immunoglobulin subclasses in mouse serum were measured using Mouse Immunoglobulin Isotyping Kit (BD Cytometric Bead Array, 550026).

Cell sorting

Peripheral lymph nodes and/or spleens were harvested from mice with indicated genotypes, and grinded into single cells, followed by treatment with Dynabeads™ Untouched™ Mouse CD4 Cells Kit (Invitrogen, 11415D) to isolate the CD4⁺ T cells. The FACS sorting (SONY Cell Sorter, SH800S) was used to isolate the CD4⁺YFP⁺ Treg cells with purities >99%.

The in vivo immune-suppression assay in Rag1 ^−/− mice

Naive T cells sorted by FACS (CD4⁺YFP^-CD44^loCD62L^hi, 4 × 10⁵) from Foxp3^cre mice were transferred into Rag1^−/− mice alone or in combination with indicated Treg cells (CD4⁺YFP⁺, 2 × 10⁵). Mice were euthanized at 4 weeks after T-cell transfer and the colon tissues were collected for histopathological analysis. The severity of colon damages were classified into five categories as described previously⁵⁸: Grade 0 has normal colonic crypt architecture; Grade 1 has mild inflammation with slight epithelial cell hyperplasia and increased numbers of leukocytes in the mucosa; Grade 2 has moderate colitis with pronounced epithelial hyperplasia, significant leukocyte infiltration, and decreased numbers of goblet cells; and Grade 3 has severe colitis with marked epithelial hyperplasia, extensive leukocyte infiltration, significant depletion of goblet cells, and occasional ulceration, or cryptic abscesses; Grade 4 has very severe colitis with marked epithelial hyperplasia, extensive transmural leukocyte infiltration, severe depletion of goblet cells, severe ulceration, and many crypt abscesses.

The in vivo adaptation of wt Treg cells

The CD4⁺YFP⁺ wt Treg cells were sorted from the Foxp3^cre mice, and transplanted to the recipient by intraperitoneal injection. For Foxp3^cre;Rbx1^fl/fl, 4 × 10⁵ wt Treg cells were transplanted to the Foxp3^cre;Rbx1^fl/fl mice at p8 stage, and the recipients were euthanized at p20 for analysis. For Foxp3^cre;Ube2m^fl/fl, 8 × 10⁵ wt Treg cells were transplanted to the Foxp3^cre;Ube2m^fl/fl mice at 6 weeks old, and the recipients were euthanized at 16-week old for analysis.

Single-cell RNA sequencing

CD4⁺YFP⁺ Treg cells were isolated from the peripheral lymph nodes of mice with indicated genotypes as above. To generate enough materials, the lymph nodes from 3 Foxp3^cre/wt or 6 Foxp3^cre/wt;Rbx1^fl/fl mice at age of 10 weeks were pooled and subjected to single-cell RNA sequencing by Sinotech Genimics Co., Ltd. (Shanghai, China). The single-cell capture was achieved by random distribution of a single-cell suspension across >200,000 microwells through a limited dilution approach, and the wt and Rbx1-deficient Treg cells were distributed on two individual microwell plates. Beads with oligonucleotide barcodes were added to saturation so that a bead was paired with a cell in a microwell. Cell-lysis buffer was added to facilitate poly-adenylated RNA molecules hybridized to the beads. Beads were collected into a single tube for reverse transcription. Upon cDNA synthesis, each cDNA molecule was tagged on the 5′ end (that is, the 3′ end of a mRNA transcript) with a molecular index and cell label to indicate its cell of origin. Whole transcriptome libraries were prepared using the BD Resolve system for single-cell whole-transcriptome amplification workflow (BD Genomics) to capture transcriptomic information of the sorted single cells. Briefly, the second strand cDNA was synthesized, followed by ligation of the adaptor for universal amplification. Eighteen cycles of PCR were used to amplify the adaptor-ligated cDNA products. Sequencing libraries were prepared using random priming PCR of the whole-transcriptome amplification products to enrich the 3′ end of the transcripts linked with the cell labels and molecular indices. Sequencing libraries were quantified using a High Sensitivity DNA chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific). Approximate 1.5 pM of the library for each sample was loaded onto a NextSeq 500 system and sequenced using High Output sequencing kits (75 × 2 bp) (Illumina). After discarding the cells with high mitochondrial gene, 10,048 and 9736 cells were identified for wt and Rbx1-deficient Treg cells, with an average number of reads of 23,321.27 vs. 22202.55, and 1080.16 vs. 870.28 of genes detected per cell, respectively.

Transcriptome profiling

CD4⁺YFP⁺ Treg cells were isolated from the peripheral lymph nodes and spleens of mice (8–12 weeks old) with indicated genotypes. To generate enough materials, the pooled tissues from 2 to 3 mice (Foxp3^cre/wt), 8 to 11 (Foxp3^cre/wt;Rbx1^fl/fl) or 3 to 4 (Foxp3^cre/wt;Ube2m^fl/fl) were used. RNA samples were prepared with the miRNeasy Mini Kit (Qiagen, 21704), then reverse transcribed, amplified, labeled (Affymetrix GeneChip pico kit, 703308), and hybridized to Clariom S Arrays, mouse (Affymetrix, 902931), performed by Cnkingbio Biotech Co., Ltd. (Beijing, China). Microarray data sets were analyzed with Applied Biosystems Expression Console Software 1.4. Three independent experiments were performed.

Proteomics profiling

CD4⁺YFP⁺ Treg cells were isolated from the peripheral lymph nodes and spleens of mice (>8 weeks of age) with indicated genotypes. To secure enough samples, tissues from 5 to 6 of Foxp3^cre/wt or 21 to 24 of Foxp3^cre/wt;Rbx1^fl/fl mice were pooled, washed with PBS buffer, and stored at −80 °C. Three independent collected pooled samples were subjected to Mass-Spectrometry.

Cells were resuspended in 5 μl 1% (w/v) sodium deoxycholate, 10 mM TCEP, 40 mM 2-chloroacetamide (CAA), 100 mM Tris (pH 8.5), and subsequently lysed by 5 min boiling at 95 °C and sonication (Bioruptor, model UCD-200, Diagenode) for 15 min. Cell debris were pelleted by centrifugation at 13,200r.p.m. for 5 min and the clarified lysate was transferred into a new vial. The lysate was diluted 1:10 for LysC-trypsin digestion (0.4 μg of each enzyme in double distilled water), and the digestion was performed overnight at 37 °C. The digest was acidified with 50 μl 2% TFA and sodium deoxycholate was extracted using 50 μl ethyl acetate and vigorous shaking. The organic phase was removed after centrifugation at 13,200r.p.m. for 5 min. Finally, the peptides were desalted on C18 StageTips.

Samples were analyzed on Orbitrap Fusion Lumos mass spectrometers (Thermo Fisher Scientific, Rockford, IL, USA) coupled with an Easy-nLC 1000 nanoflow LC system (Thermo Fisher Scientific). Dried peptide samples were re-dissolved in Solvent A (0.1% formic acid in water) and loaded to a trap column (100 μm × 2 cm, home-made; particle size, 3 μm; pore size, 120 Å; Dr. Maisch, Ammerbuch, Germany) with a max pressure of 280 bar using Solvent A, then separated on a home-made 150 μm × 30 cm silica microcolumn (particle size, 1.9 μm; pore size, 120 Å; Dr. Maisch, Ammerbuch, Germany) with a gradient of 5–35% mobile phase B (acetonitrile and 0.1% formic acid) at a flow rate of 600 nl/min for 141 min then up to 95% in 1 min and eluted for 9 min (the column temperature was maintained at 60 °C). The MS analysis were performed in data-dependent acquisition mode. Automatic gain control (AGC) targets were 5 × 10⁵ ions with a max injection time of 50 ms for full scans and 5 × 10³ with 35 ms for MS/MS scans. The most intense ions selected under top-speed mode were isolated in Quadrupole with a 1.6 m/z window and fragmented by higher energy collisional dissociation (HCD) with normalized collision energy of 32% and dynamic exclusion time was set as 25 s. Data were acquired using the Xcalibur software (Thermo Fisher Scientific).

DAVID was used for pathway enrichment analysis of proteins with change fold >2/3 or <0.5/0.33. The enriched pathways and their contained proteins with two-fold change were shown in heat map in which each quantitative value was normalized based its maximum value.

RNA and immunoblot analysis

The sequences of the primer pairs used in qPCR are as follows: Sag-Fwd: 5′-TG GAGGACGGCGAGGAAC-3′, Sag-Rev: 5′-CCCCAGACCACAACACAGTC-3′; Rbx1-Fwd: 5′-CTTTGTATCGAATGTCAGGC-3′, Rbx1-Rev: 5′-GTCACTAGACGAGTAACAG-3′; Ube2f-Fwd: 5′-GACTGTAAGCCCAGATGAG-3′, Ube2f-Rev: 5′-CCTTTAATGTTCTAGTGGG-3′; Ube2m-Fwd: 5’-CTGTCCTGATGAAGGCTTC-3′, Ube2m-Rev: 5′-GTTCGGCTCCAAGAAGAG-3′; Gapdh-Fwd: 5′-GCCGCCTGGAGAAACCTGCC-3′, Gapdh-Rev: 5′-GGTGGAAGAGTGGGAGTTGC-3′.

The antibodies used in western blot are as follows: anti-Ube2f, Proteintech, 17056-1-AP (dilution 1: 1000); and anti-β-Actin, HuaBio, M1210-2 (dilution 1: 5000).

Statistical analysis

The p values were calculated by Mann–Whitney test, two-sided unpaired Student’s t-test, one-way ANOVA or two-way ANOVA as indicated using GraphPad Prism, unless otherwise noted. Statistical analysis of mouse survival and respective p values were determined using the log-rank test. p < 0.05 was considered as significant. All error bars represent the SEM from three independent experiments.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The scRNA-seq and microarray data generated in this study have been deposited in the Gene Expression Omnibus under accession code GSE199289 and GSE192664. The mass spectrometry proteomics data generated in this study have been deposited in the ProteomeXchange Consortium via the iProX partner repository⁷³ under accession code PXD030144. Source data are provided with this paper.

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Acknowledgements

We would like to thank Prof. Xiaoming Feng for providing Foxp3^YFP-cre mice. We also thank the Core Facilities, Zhejiang University School of Medicine for the technical support. This project was supported by the National Key R&D Program of China (2021YFA1101000 and 2016YFA0501800 to Y.S.), National Natural Science Foundation of China (82172699 and 81801567 to D.W.), and Zhejiang Provincial Natural Science Foundation of China (LY21H100005 to D.W. and LD22H300003 to Y.S.).

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Authors and Affiliations

Cancer Institute of the Second Affiliated Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310029, China
Di Wu & Yi Sun
Cancer Center, Zhejiang University, Hangzhou, Zhejiang, 310058, China
Di Wu & Yi Sun
Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, Zhejiang, 310053, China
Di Wu & Yi Sun
Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310003, China
Haomin Li
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing) and Institute of Lifeomics, Beijing, 102206, China
Mingwei Liu & Jun Qin

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Di Wu
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Haomin Li
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Mingwei Liu
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Jun Qin
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Yi Sun
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Contributions

D.W. designed and preformed experiments, analyzed data, and wrote the manuscript. H.L. analyzed proteome and transcriptome data. M.L. performed the mass spectrum experiment and analyzed the data. J.Q. designed and supervised the mass spectrum experiment and analyzed data. Y.S. designed experiments, analyzed data, wrote and finalize the manuscript, and oversaw the project.

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Correspondence to Yi Sun.

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Wu, D., Li, H., Liu, M. et al. The Ube2m-Rbx1 neddylation-Cullin-RING-Ligase proteins are essential for the maintenance of Regulatory T cell fitness. Nat Commun 13, 3021 (2022). https://doi.org/10.1038/s41467-022-30707-8

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Received: 05 October 2020
Accepted: 12 May 2022
Published: 31 May 2022
DOI: https://doi.org/10.1038/s41467-022-30707-8

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