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American Association for the Advancement of Science

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10.1126/sciadv.ado4274
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Immunology
Cancer-associated SF3B1-K700E mutation controls immune responses by regulating Treg function via aberrant Anapc13 splicing
SF3B1-K700E mutation impairs the Treg function
https://orcid.org/0000-0002-4114-3388
Shi Yun Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Supervision Validation Visualization Writing - original draft Writing - review & editing 1
https://orcid.org/0000-0002-8828-2000
Zhang Wencan Investigation Methodology 1
https://orcid.org/0000-0002-3063-2346
Jia Qiong Data curation Formal analysis Software Visualization 2
https://orcid.org/0000-0001-8312-4057
Zhong Xiancai Investigation Methodology Visualization 1
https://orcid.org/0000-0001-6982-6343
Iyer Prajish Resources 3
https://orcid.org/0000-0002-0762-8322
Wu Hongmin Methodology 1
https://orcid.org/0000-0002-3698-9784
Yuan Yate-Ching Conceptualization Data curation Formal analysis Methodology Project administration Software Validation Visualization Writing - review & editing 4
https://orcid.org/0000-0002-4256-4512
Zhao Yuqi Formal analysis Software 5
Zhang Lianjun Methodology 6
https://orcid.org/0000-0001-5028-2868
Wang Lili Methodology Resources Writing - review & editing 3
https://orcid.org/0000-0002-1428-904X
Jia Zhenyu Formal analysis Software Validation Visualization 2
https://orcid.org/0000-0003-2595-0419
Kuo Ya-Huei Methodology Project administration Resources Supervision Writing - original draft Writing - review & editing 6
https://orcid.org/0000-0003-1896-6666
Sun Zuoming Conceptualization Funding acquisition Methodology Project administration Supervision Validation Writing - original draft Writing - review & editing 1 *
1 Department of Immunology & Theranostics, Arthur Riggs Diabetes & Metabolism Research Institute, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
2 Department of Botany & Plant Sciences, University of California, Riverside, CA 92527, USA.
3 Department of System Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
4 Translational Bioinformatics, Department of Computational Quantitative Medicine, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
5 Integrated Genomics Core, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
6 Gehr Family Center for Leukemia Research, Department of Hematological Malignancies Translational Science, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
* Corresponding author. Email: zsun@coh.org
20 9 2024
20 9 2024
10 38 eado427401 2 2024
14 8 2024
Copyright © 2024 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).
2024
The Authors
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Recurrent somatic mutations in spliceosome factor 3b subunit 1 (SF3B1) are identified in hematopoietic malignancies, with SF3B1-K700E being the most common one. Here, we show that regulatory T cell (Treg)–specific expression of SF3B1-K700E (Sf3b1K700Efl/+/Foxp3YFP-Cre) results in spontaneous autoimmune phenotypes. CD4+ T cells from Sf3b1K700Efl/+/Foxp3YFP-Cre mice display defective Treg differentiation and inhibitory function, which is demonstrated by failed prevention of adoptive transfer colitis by Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs. Mechanically, SF3B1-K700E induces an aberrant splicing event that results in reduced expression of a cell proliferation regulator Anapc13 due to the insertion of a 231–base pair DNA fragment to the 5′ untranslated region. Forced expression of the Anapc13 gene restores the differentiation and ability of Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs to prevent adoptive transfer colitis. In addition, acute myeloid leukemia grows faster in aged, but not young, Sf3b1K700Efl/+/Foxp3YFP-Cre mice compared to Foxp3YFP-Cre mice. Our results highlight the impact of cancer-associated SF3B1 mutation on immune responses, which affect cancer development.

SF3B1-K700E mutation controls immune responses via alternative Anapc13 splicing to regulate Treg function.

http://dx.doi.org/10.13039/100000002 National Institutes of Health NIH R01-AI109644 http://dx.doi.org/10.13039/100000002 National Institutes of Health R21-AI163256 http://dx.doi.org/10.13039/100000002 National Institutes of Health P30CA033572 Jackie and Bruce Barrow Cancer Research Scholars’Program AR-DMRI Innovative grant Caltech-CoH Biomedical Initiative
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pmcINTRODUCTION

Spliceosome factor 3b subunit 1 (SF3B1), a critical component of the core spliceosome U2 small nuclear ribonucleoprotein complex, regulates pre-mRNA splicing (1–3). Large-scale cancer genome sequencing projects have identified recurrent somatic mutations in SF3B1 in several types of hematological malignancies including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS) (4–6). About half of MDS tumor samples have mutations in spliceosome genes, with SF3B1 being the most commonly mutated one (7, 8). Several lines of evidence support that SF3B1 mutations often represent founding genetic lesions and thus are major determinants of disease phenotype and have independent prognostic values on survival and risk of progression to AML (6, 9–12). The most common SF3B1 mutation is an A to G transition that results in lysine to glutamic acid substitution at amino acid position 700 (SF3B1-K700E) (4, 13). Previous studies have focused on how mutant SF3B1 intrinsically promotes the development of cancers via aberrant RNA splicing. For example, it has been demonstrated that the SF3B1-K700E mutation alters splicing events, resulting in the dysregulation of multiple cellular pathways including DNA damage and telomere maintenance pathways that drive oncogenesis (14–16). However, SF3B1 mutations can be traced back to earlier hematopoietic cells that differentiate into different types of immune cells including regulatory T cells (Tregs). It largely remains unknown how SF3B1 mutation affects the function of Tregs that plays an important role in controlling the scale of immune responses.

Tregs are required to protect against autoimmune responses, maintain homeostasis, and dampen immune responses after clearance of infection (17). The important physiological function of Tregs for induction and maintenance of peripheral tolerance is illustrated by the uncontrollable autoinflammation in mice and humans that lack functional Tregs due to a mutation in the Forehead Box P3 (Foxp3) gene (18–20). Foxp3 is a lineage-specific transcription factor that regulates the generation, maintenance, and function of Tregs (21). Natural Tregs (nTregs) develop in the thymus (or called tTregs) mostly with T cell receptors recognizing self-antigens (22, 23), whereas induced Tregs (iTreg) are differentiated from activated naive CD4+ T cells in the presence of transforming growth factor–β (TGF-β) (21, 24). In addition to Treg, naive CD4+ T cells also differentiate into inflammatory T cells including T helper 1 (TH1), TH2, and TH17 (25, 26), which are inhibited by Tregs. A fine-tuned balance between inflammatory T cells and Tregs is essential for a functional immune system. Skewing to inflammatory T cells leads to autoimmunity, whereas immune tolerance is induced by the dominance of Tregs. Thus, understanding the mechanisms that regulate the differentiation and function of Tregs facilitates the development of novel immunotherapies for controlling immune responses.

Considering the importance of Tregs in controlling immune responses, we determined how hematological malignancy-associated Sf3b1 mutation, Sf3b1-K700E in this case, affects the Tregs by using mice that express Sf3b1-K700E specifically in Tregs (Sf3b1K700Efl/+/Foxp3YFP-Cre). Sf3b1K700Efl/+/Foxp3YFP-Cre mice displayed autoimmune phenotypes including splenomegaly and infiltration of lymphocytes including interferon-γ (IFN-γ)–producing T cells to tissues such as lung and liver. Upon induction, Sf3b1K700Efl/+/Foxp3YFP-Cre mice developed aggravated experimental autoimmune encephalomyelitis (EAE), which was associated with a reduced number of Tregs. CD4+ T cells were defective in Treg differentiation. In addition, Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs had greatly impaired inhibitory function in vitro in inhibiting T cell activation and in vivo in preventing colitis induced by adoptive transfer of naive CD4+ T cells. We identified an aberrant splicing event at the Anapc13 gene induced by Sf3b1-K700E expression that accounted for the impaired Treg differentiation and function observed in Sf3b1K700Efl/+/Foxp3YFP-Cre mice. The Sf3b1-K700E–induced aberrant splicing event resulted in the insertion of a DNA fragment to the 5′ untranslated region (5′UTR) of the Anapc13 gene, which greatly reduced Anapc13 expression. On the other hand, forced expression of Anapc13 in Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs restored the differentiation and function of Tregs. In addition, transplanted AML cells grew faster in aged, but not young, Sf3b1K700Efl/+/Foxp3YFP-Cre mice. Our results thus highlight the vital impact of this hematological malignancy-associated mutation on immune tolerance and cancer development.

RESULTS

Sf3b1-K700E mutant impairs Treg differentiation

To determine whether and how Sf3b1-K700E mutation affects Tregs, we used knock-in Sf3b1K700Efl/+ mice in which Cre induces the expression of Sf3b1-K700E from the chromosome containing the knock-in allele, whereas the wild-type (WT) Sf3b1 is expressed from the other chromosome (14, 27), as such mutation is often heterozygous. Sf3b1K700Efl/+ mice were crossed to Foxp3YFP-Cre mice or CD4Cre mice to express Sf3b1-K700E only in Tregs (Sf3b1K700Efl/+/Foxp3YFP-Cre mice) or T cells (Sf3b1K700Efl/+/CD4Cre mice) (fig. S1A for genotyping WT and knock-in allele). Sequencing analysis confirmed K700 (AAA) to E (GAA) mutation (fig. S1B). Two peaks observed at the mutated nucleotide indicate the mixture of A from the WT chromosome and mutated nucleotide G from the chromosome containing the knock-in allele.

Since Tregs develop in the thymus (tTregs), thymic Treg development was first examined. Thymocyte development was overall normal in Sf3b1K700Efl/+/Foxp3YFP-Cre mice, indicated by equivalent thymic cellularity (fig. S1C) and percentage of different developmental stages of thymocyte subsets: CD4−CD8− double-negative (early thymocytes), CD4+CD8+ double-positive, and CD4+/CD8+ single-positive (mature T cells) cells when compared to Foxp3YFP-Cre mice (fig. S1D). There was no notable difference in the percentage and the number of thymic Tregs between Foxp3YFP-Cre and Sf3b1K700Efl/+/Foxp3YFP-Cre mice (Fig. 1, A and B). We further monitored Helios and Nrp-1 expression that was reported to be up-regulated on tTregs (28, 29) and did not observe the obvious difference in their expression on Tregs from Foxp3YFP-Cre and Sf3b1K700Efl/+/Foxp3YFP-Cre spleens (fig. S1E). Thus, our results do not support that Sf3b1-K700E mutation obviously affects Treg development in the thymus.

Fig. 1. Sf3b1-K700E mutant impairs Treg differentiation.

(A and B) Representative flow cytometric analysis [(A), left], the percentage [(A), right], and number (B) of Treg (Foxp3+) cells in thymocytes from indicated mice (n ≥ 4 per genotype). (C and D) Representative flow cytometric analysis [(C), left] and percentage [(C), right] of Foxp3+ Tregs differentiated from Foxp3YFP-Cre or Sf3b1K700EFl/+/Foxp3YFP-Cre naive CD4+ T cells stimulated in the presence of TGF-β (5 ng/ml) for 48 hours (n ≥ 4 per treatment cohort). Foxp3 mRNA levels in differentiated Tregs shown in (C) were detected by quantitative polymerase chain reaction (qPCR) (D). (E and F) Representative flow cytometric analysis [(E), left] and percentage [(E), right] of Foxp3+ Tregs differentiated from naive WT, Sf3b1K700EFl/+, and Sf3b1K700EFl/+/CD4Cre CD4+ T cells stimulated in the presence of TGF-β ( 5 ng/ml) for 48 hours (n ≥ 4 per treatment cohort). Foxp3 mRNA levels in differentiated Tregs shown in (E) were detected by qPCR (F). (G) Representative flow cytometric analysis (left) and percentage (right) of the proliferating Foxp3YFP-Cre or Sf3b1K700EFl/+/Foxp3YFP-Cre CD4+ T cells in indicated peak shown on left, labeled with CellTrace Violet (CTV), polarized under Treg conditions for 48 hours (n ≥ 4 per genotype). (H) Representative flow cytometric analysis (left) and percentage (right) of the proliferative dye–labeled WT, Sf3b1K700EFl/+, and Sf3b1K700EFl/+/CD4Cre CD4+ T cells in indicated peak shown on left, labeled with CTV, polarized under Treg conditions for 48 hours (n ≥ 4 per genotype). Boxed area: cell population of interest. Data are from three experiments [(B), (D), and (F); (A), (C), (E), and (G) to (I), right panels, presented as means ± SEM] or are from one representative of three independent experiments [(A), (C), (E), (G), and (H), left panels]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0005; ns, not significant (two-tailed Student’s t test).

iTregs are differentiated from peripheral naive CD4+ T cells in the presence of TGF-β. We next examined how Sf3b1-K700E mutation affects iTreg differentiation. We confirmed that purified naive CD4+ T cells from spleens of Foxp3YFP-Cre and Sf3b1K700Efl/+/Foxp3YFP-Cre mice were Foxp3− (fig. S1F), whereas these naive CD4+ T cells differentiated into Foxp3+ Tregs when activated in the presence of TGF-β (Fig. 1C). However, the ability of CD4+ T cells from Sf3b1K700Efl/+/Foxp3YFP-Cre mice to generate iTregs was impaired compared to the CD4+ T cells from Foxp3YFP-Cre mice. Consistently, Foxp3 mRNA was decreased in Sf3b1K700Efl/+/Foxp3YFP-Cre CD4+ T cells compared to that of Foxp3YFP-Cre CD4+ T cells after Treg differentiation (Fig. 1D). The observed impaired Treg differentiation was not due to changes in cell survival, which were comparable between Foxp3YFP-Cre and Sf3b1K700Efl/+/Foxp3YFP-Cre cells (fig. S1G). The impaired iTreg differentiation together with reduced Foxp3 mRNA was also confirmed using naive CD4+ T cells (fig. S1H) from Sf3b1K700Efl/+/CD4Cre mice that displayed more marked defects than that observed with Sf3b1K700Efl/+/Foxp3YFP-Cre CD4+ T cells (Fig. 1, E and F). This is likely due to that the expression of the Sf3b1-K700E mutant in Sf3b1K700Efl/+/CD4Cre CD4+ T cells was before the induction of Treg differentiation, whereas Sf3b1K700Efl/+/Foxp3YFP-Cre CD4+ T cells started to express the Sf3b1-K700E mutant after Treg differentiation when Foxp3 was induced. We next monitored cell proliferation during Treg differentiation and found that the proliferation of Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs was slower than that of Foxp3YFP-Cre Tregs (Fig. 1G). Consistently, reduced proliferation was also observed in Sf3b1K700Efl/+/CD4Cre Tregs (Fig. 1H). However, the defective proliferation was not observed in stimulated purified Tregs (fig. S1I). Therefore, the Sf3b1-K700E mutant impairs the differentiation of iTregs by reducing proliferation.

Sf3b1-K700E mutant impairs the generation of iTreg in vivo

To determine whether the Sf3b1-K700E mutant affects the Treg differentiation in vivo, sorted naive Foxp3YFP-Cre or Sf3b1K700Efl/+/Foxp3YFP-Cre CD4+ T cells that lack Foxp3+ Tregs (fig. S1F) were adoptively transferred to Rag1−/− mice (30, 31). Tregs were detected in spleens and mesenteric lymph nodes (mLNs) 3 weeks after the adoptive transfer of naive Foxp3YFP-Cre CD4+ T cells (Fig. 2A). However, naive Sf3b1K700Efl/+/Foxp3YFP-Cre CD4+ T cells generated much fewer Tregs in vivo. Next, an oral tolerance model was used to determine the effects of Sf3b1-K700E on the generation of Tregs in vivo (24). In this model, sorted naive CD4+ T cells from OT-II/Sf3b1K700Efl/+ or OT-II/ Sf3b1K700Efl/+/CD4Cre mice were adoptively transferred into Rag1−/− mice, and a notable amount of Tregs was induced mostly in gut-associated lymphoid tissues such as the colon and mLNs, but not the spleen, by orally administrated ovalbumin (OVA) peptide in drinking water (Fig. 2B). Consistently, much less Tregs were induced from OT-II/Sf3b1K700Efl/+/CD4Cre CD4+ T cells than that from OT-II/Sf3b1K700Efl/+ CD4+ T cells. Collectively, these results demonstrate that the Sf3b1-K700E mutant impairs Treg differentiation in vivo.

Fig. 2. Sf3b1-K700E mutant impairs the generation of iTreg in vivo.

(A) Representative flow cytometric analysis (left) and percentage (right) of Foxp3+CD4+ Tregs in the colon and mesenteric lymph nodes (mLNs) 3 weeks after adoptive transfer of indicated 0.4 × 106 naive CD4+ T cells to Rag1−/− mice (n ≥ 4 per genotype). (B) Representative flow cytometric analysis (left) and percentage (right) of Foxp3+CD4+ Tregs in colon, mLN, and spleen of Rag1−/− mice transferred with 3 × 106 naive OT-II/ Sf3b1K700Efl/+ or OT-II/ Sf3b1K700Efl/+/CD4Cre naive CD4+ T cells and subsequently fed with ovalbumin (OVA)-containing drinking water (20 mg/ml) for 5 days (n ≥ 4 per genotype). (C) Mean clinical EAE scores of indicated mice at different days after EAE induction with MOG35–55 (n = 9 per genotype). (D and E) Representative flow cytometric analysis [(D), left], the percentage [(D), right], and number (E) of Foxp3+CD4+ Tregs recovered from the central nervous system (CNS) of EAE-induced mice shown in (C) (n ≥ 5 per genotype). (F) Representative flow cytometric analysis (left) and the percentage (right) of IFN-γ+ and interleukin-17A–positive (IL-17A+) cells among CD4+ T cells recovered from the CNS of EAE-induced mice shown in (C) (n ≥ 7 per genotype). (G) Section of hematoxylin and eosin (H&E)–stained spinal cord from post-EAE induction in indicated mice shown in (C) (left). The right panel is the inflammation score based on observed lymphocyte infiltration shown on the left panels (n ≥ 4 per genotype). Boxed area: cell population of interest. Data are from three experiments [(D), presented as means ± SEM; (B), (C), (E), and (G), right panels, presented as means ± SEM] or are from one representative of three independent experiments [(A); (B), (C), (E), and (G), left panels]. *P < 0.05 (two-tailed Student’s t test).

To determine whether the Sf3b1-K700E mutant–impaired generation of Tregs affects immune responses in vivo, we compared the development of EAE between Foxp3YFP-Cre and Sf3b1K700Efl/+/Foxp3YFP-Cre mice. Compared to Foxp3YFP-Cre mice, Sf3b1K700Efl/+/Foxp3YFP-Cre mice developed much severe EAE together with more weight loss (Fig. 2C and fig. S2A) and had less number and percentage of Tregs (Fig. 2, D and E), whereas more inflammatory CD4+IFN-γ+ and CD4+IL-17A+ (interleukin-17A–positive) cells in the central nervous system (CNS) (Fig. 2F and fig. S2B for gating strategy). Consistently, the histochemical examination also observed that Sf3b1K700Efl/+/Foxp3YFP-Cre mice had more infiltrated lymphocytes to CNS and tissue damages with a much higher inflammation score (Fig. 2G). Our results thus demonstrate that the Sf3b1-K700E mutant impairs Treg differentiation in vivo, which leads to aggravated immune responses for induction of EAE.

Tregs from Sf3b1K700Efl/+/Foxp3YFP-Cre mice have impaired inhibitory function

In addition to Treg differentiation, the ability of Treg to suppress T cell activation also controls the scale of immune responses. We first examined the expression of several surface markers, CD25, CD73, CD39, and CTLA-4 (fig. S3A) that are the indicators for the suppressive function of Tregs (32–34). Sf3b1K700Efl/+/Foxp3YFP-Cre Treg had lower levels of CD25, CD73, and Foxp3, already indicating impaired suppressive function. We next assessed the ability of Tregs to suppress CD4+ T cell proliferation in vitro. In vitro differentiated iTregs from Sf3b1K700Efl/+/Foxp3YFP-Cre mice showed greatly impaired suppressive function in inhibiting CD4+ T cell proliferation compared to the Tregs from Foxp3YFP-Cre mice (Fig. 3A and fig. S3B). We also sorted YFP+Nrp-1− iTreg directly from mice to determine their ability to suppress CD4+ T cell proliferation (fig. S3C). Consistently, iTreg from Sf3b1K700Efl/+/Foxp3YFP-Cre mice had reduced inhibitory activity compared to iTreg from Foxp3YFP-Cre mice. Next, the in vivo suppressive function of Tregs was examined in the prevention of adoptive transfer colitis. In the absence of Tregs, adoptive transfer of naive CD4+ T cells (CD45RBhiCD25−CD4+) into Rag1−/− mice induced severe colitis, as indicated by weight loss (Fig. 3B), shortened colon (Fig. 3C), damaged tissues (Fig. 3D), and greatly increased pro-inflammatory IFN-γ+CD4+ T, but not IL-17A+CD4+, cells in the colon and mLN (Fig. 3E), whereas cotransfer of Foxp3YFP-Cre Tregs, but not Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs, prevented these severe colitis phenotypes in Rag1−/− mice (Fig. 3, B to E). Furthermore, a lower percentage of Tregs were detected in the recipients adoptively transferred with Sf3b1K700Efl/+/Foxp3YFP-Cre Treg compared to the recipients with Foxp3YFP-Cre Tregs in spleen and gut-associated tissues including colon and mLN (Fig. 3F), thus contributing to the observed much severe colitis. Similarly, adoptive transfer of Tregs from Sf3b1K700Efl/+/CD4Cre mice also failed to prevent colitis to the levels by Tregs from control Sf3b1K700Efl/+ mice, indicated by more weight loss (fig. S3D), a shorter colon (fig. S3E), and a higher number of pro-inflammatory IFN-γ+CD4+ T, but not IL-17A+CD4+, cells recovered from the colon (fig. S3F), whereas there were fewer Tregs in the spleen, colon, and mLN in Rag1−/− recipients with Sf3b1K700Efl/+/CD4Cre Tregs (fig. S3G). Their results suggest that the Sf3b1-K700E mutant impairs the suppressive function of Tregs in addition to Treg differentiation.

Fig. 3. Tregs from Sf3b1K700Efl/+/Foxp3YFP-Cre mice have impaired inhibitory function.

(A) Representative flow cytometric analysis (left) and percentage (right) of the proliferated responder CD4+ T cells (Tresp) in the gated area shown on left, labeled with CTV dye and cocultured with splenic YFP+CD4+ Tregs isolated from indicated 6- to 8-week-old mice (n ≥ 4 per genotype). (B) Body weight of Rag1−/− recipients over time after adoptive transfer of naive WT CD45RBhiCD25−CD4+ T cells alone or in combination with purified splenic Tregs from indicated 6- to 8-week-old mice. (C) Representative image of colons (left) and colon length (right) (n ≥ 5 per genotype) from colitis-induced mice shown in (B). (D) H&E-stained colon section from colitis-induced recipients shown in (B) 9 weeks after adoptive transfer. (E) Representative flow cytometric analysis (left) and percentage (right) of CD4+IL-17A+ and CD4+IFN-γ+ cells recovered from colons or mLN of colitis-induced recipients shown in (B) (n ≥ 4 per group). (F) Representative flow cytometric analysis (left) and percentage (right) of Foxp3+CD4+ Tregs recovered from colon, spleen, and mLN of colitis-induced recipients shown in (B) (n ≥ 4 per group). Boxed area: cell population of interest. Data are from three experiments [(A) to (C), (E), and (F), right panels, presented as means ± SEM] or are from one representative of three independent experiments [(B); (A), (C), (D), and (F), left panels]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0005 (two-tailed Student’s t test).

Sf3b1K700Efl/+/Foxp3YFP-Cre mice develop autoinflammation

We noticed that aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice were smaller indicated by the lighter weight (Fig. 4A) and suffered from dermatitis associated with hair loss (fig. S4A) compared to their Foxp3YFP-Cre counterparts. Histochemical analysis of the skin found notable lymphocyte infiltration in the skin in Sf3b1K700Efl/+/Foxp3YFP-Cre mice (fig. S4B). Sf3b1K700Efl/+/Foxp3YFP-Cre mice had enlarged spleen compared to Foxp3YFP-Cre mice (Fig. 4B, left), and the difference in spleen size was increased with aging (Fig. 4B, right). The increased spleen size was also reflected by increased spleen weight (Fig. 4C) and cellularity (Fig. 4D) including CD3+ T cells due to an increase in both CD4+ and CD8+ T cells (Fig. 4E, left). In addition, lymph nodes in Sf3b1K700Efl/+/Foxp3YFP-Cre mice were also larger, indicated by increased cellularity (Fig. 4E, right). These phenotypes indicate that Sf3b1K700Efl/+/Foxp3YFP-Cre mice develop spontaneous autoinflammation. Consistently, analysis of CD4+ T cells indicated that there were more CD44hiCD62Llo memory-like, whereas there were reciprocally less CD44loCD62Lhi naive cells both in 8- to 10-week-old (fig. S4C) and 30- to 35-week-older Sf3b1K700Efl/+/Foxp3YFP-Cre mice compared to Foxp3YFP-Cre mice (Fig. 4F). Furthermore, more IFN-γ+, but not IL-17A+, inflammatory CD4+ T cells were detected in spleens and lymph nodes of older Sf3b1K700Efl/+/Foxp3YFP-Cre mice than that of Foxp3YFP-Cre mice (Fig. 4G). Increased IFN-γ+ CD4+ T cells were also observed in younger Sf3b1K700Efl/+/Foxp3YFP-Cre mice (fig. S4D). Histological analysis of lung and liver found notable lymphocyte infiltration together with tissue damage in Sf3b1K700Efl/+/Foxp3YFP-Cre mice (Fig. 4H), similar to that observed in the skin (fig. S4B). Increased percentage and/or number of Tregs were observed in the spleen, lymph node, lung, and liver of Sf3b1K700Efl/+/Foxp3YFP-Cre mice (Fig. 4I and fig. S4, E and F). However, these Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs had substantially reduced mean fluorescence intensity for Foxp3 compared to Foxp3YFP-Cre Tregs (fig. S4G). The increased Tregs in Sf3b1K700Efl/+/Foxp3YFP-Cre mice are likely a compensatory mechanism trying to inhibit the observed inflammation. This is consistent with what was observed in other mice with defective Tregs (35, 36). These results suggest that Treg-specific expression of the Sf3b1-K700E mutant breaks the immune tolerance and leads to the development of autoinflammation.

Fig. 4. Sf3b1K700Efl/+/Foxp3YFP-Cre mice develop autoinflammation.

(A) Body weight of indicated 8- to 10-week-old or 30- to 35-week-old mice (n ≥ 5 per genotype per group). (B) Image of spleens from indicated 8- to 10-week-old (left) or 30- to 35-week-old mice (right). (C) Weight of the spleens from indicated 8- to 10-week-old or 30- to 35-week-old mice (n ≥ 4 per genotype per group). (D and E) Total number of cells (D) and CD3+, CD4+, and CD8+ T cells [(E), left] of spleens or inguinal lymph nodes (iLN) or mLNs [(E), right] from indicated mice (n ≥ 4 per genotype per group). (F) Percentage of CD44hiCD62lo memory-like (left) and CD44loCD62hi (right) naive cells among splenic CD4+ T cells from indicated aged mice (n ≥ 4 per genotype per group). (G) Representative flow cytometric analysis (left) and percentage (right) of IL-17A+ and IFN-γ+ cells among CD4+ T cells from spleens or mLN of indicated aged mice (n ≥ 4 per genotype). (H) Section of H&E-stained lung and liver from indicated 30- to 35-week-old mice. (I) Representative flow cytometric analysis (left) and percentage (right) of Tregs (Foxp3+) among CD4+ T cells recovered from lung and liver of 30- to 35-week-old mice (n ≥ 4 per genotype). Boxed area: cell population of interest. Data are from three experiments [(A) and (C) to (F); (G) and (I), right panels, presented as means ± SEM] or are from one representative of three independent experiments [(B) and (H); (G) and (I), left panels].*P < 0.05, **P < 0.01, and ***P < 0.001 (two-tailed Student’s t test).

Sf3b1-K700E mutant induced aberrant splicing events during Treg differentiation

We next determined mechanisms for Sf3b1-K700E–impaired Treg differentiation and function. We first excluded the possibility that Foxp3 stability is affected, as the degradation rate of Foxp3 between Foxp3YFP-Cre and Sf3b1K700Efl/+/Foxp3YFP (fig. S5A) or between Sf3b1K700Efl/+ and Sf3b1K700Efl/+/CD4Cre (fig. S5B) Tregs was equivalent. SF3B1 is a splicing factor, and Sf3b1-K700E mutation is known to affect cellular function by inducing aberrant splicing events to alter gene expression (37). Thus, we next performed deep RNA sequencing (RNA-seq) analysis to detect differential splicing events and transcriptomes between Sf3b1K700Efl/+ and Sf3b1K700Efl/+/CD4Cre Tregs. Since Sf3b1K700Efl/+/CD4-Cre cells displayed more marked defects in Treg differentiation compared to Sf3b1K700Efl/+/Foxp3YFP-Cre cells (Fig. 1, E and F), Sf3b1K700Efl/+/CD4Cre cells were used in RNA-seq assay to capture the maximum differences in gene expression. The expression of the Sf3b1-K700E mutant was detected in all three RNA samples prepared from Sf3b1K700Efl/+/CD4Cre but not Sf3b1K700Efl/+ Tregs, confirming Cre-induced expression of Sf3b1-K700E (Fig. 5A). Consistent with the observed impaired Treg differentiation of Sf3b1K700Efl/+/CD4Cre cells, Foxp3, Id1, Irf4, and Myb, which are known to positively regulate Treg differentiation (24, 38–40), were among the 30 down-regulated genes in these cells (Fig. 5, B and C) that were also confirmed by individual quantitative polymerase chain reaction (qPCR) analysis (Fig. 5D). However, forced expression of Irf3, Myb, or Atf3 did not rescue Treg differentiation in Sf3b1K700Efl/+/CD4Cre CD4+ T cells (fig. S5C). By alignment of genome DNA and coding sequence derived from RNA-seq, 2382 alternative splicing events in 1414 genes were identified. There were generally five different types of alternative splicing events, alternative 3′ splice site (A3SS), alternative 5′ splice site (A5SS), skipped exon, mutually exclusive exons, and retained intron (Fig. 5E). It is consistent with multiple reports that Sf3b1 mutants induce a high frequency of aberrant A3SS (37, 41, 42). Previous studies have identified a motif associated with canonical 3′ splice sites (3SSs) in cells expressing WT SF3B1 and a different motif associated with A3SS in cells expressing mutant SF3B1 (37, 41–43). Our analysis of the sequence surrounding 3SS confirmed the motif associated with canonical 3SS in Sf3b1K700Efl/+cells expressing WT SF3B1 and a different motif associated with A3SS in Sf3b1K700Efl/+/CD4Cre cells expressing the Sf3b1-K700E mutant (Fig. 5F). These results indicate that Sf3b1-K700E induces aberrant splicing events in Tregs. We next cross-examined 61 differentially expressed genes (Fig. 5B) and 1414 alternatively spliced genes to determine which differentially expressed genes are alternatively spliced and identified anaphase-promoting complex subunit 13 (Anapc13) (Fig. 5G). Anapc13 was among the down-regulated genes in Sf3b1K700Efl/+/CD4Cre Tregs (Fig. 5C), which was confirmed by individual qPCR analysis (Fig. 5H). Anapc13 protein levels were also down-regulated in Sf3b1K700Efl/+/CD4Cre Tregs (Fig. 5I). Anapc13 is known to regulate cell proliferation as deletion of this gene stalls cell cycle progression (44). Sf3b1K700Efl/+/CD4Cre Tregs, which expressed lower levels of Anapc13 (Fig. 5, H and I), displayed reduced proliferation compared to the control Sf3b1K700Efl/+ cells (Fig. 1H). Therefore, our RNA-seq analysis indicates that Sf3b1-K700E induces aberrant splicing events including the Anapc13 gene that is known to promote cell cycle progression but is down-regulated in slower proliferative Sf3b1K700Efl/+/CD4Cre Tregs.

Fig. 5. Sf3b1-K700E mutant induced aberrant splicing events during Treg differentiation.

(A) Sequence fragment density around Sf3b1-K700E from RNA-seq performed with iTreg differentiated from naive Sf3b1K700Efl/+/CD4Cre and Sf3b1K700Efl/+ CD4+ T cells in the presence of TGF-β (5 ng/ml) for 48 hours. Red denotes WT (adenine) and blue denotes mutant (guanine) nucleotide reads. Mutant allele frequency ranged from 22.9 to 38.7%. (B) Venn diagram of a number of differentially expressed genes between Sf3b1K700Efl/+ and Sf3b1K700Efl/+/CD4Cre iTreg (n = 3 per genotype). Up-regulated genes (red) and down-regulated genes (blue) in Sf3b1K700Efl/+/CD4Cre iTreg with a cutoff at P < 0.05 and fold change |FC| ≥ 1.5 are shown. (C) A list of down-regulated genes in Sf3b1K700Efl/+/CD4Cre iTreg cells. (D) qPCR analysis of Foxp3, Atf3, Id1, Myb, and Irf4 mRNA in indicated iTreg cells (n ≥ 4 per genotype per group). (E) The number and indicated types of alternative splicing events in Sf3b1K700Efl/+/CD4Cre iTreg cells, identified by the alignment of RNA-seq sequence with genomic DNA sequence (three mice per group; false discovery rate < 0.1). (F) Motif frequency plots for canonical and aberrant 3′ splice site (3SS). The motifs showing are 35 nucleotides (nt) upstream of the 30 AG and 2 nt downstream. (G) Venn diagram of gene overlapping between 61 differentially expressed genes and 1414 alternative splicing genes between Sf3b1K700Efl/+ and Sf3b1K700Efl/+/CD4Cre iTreg. (H) qPCR analysis of Anapc13 mRNA levels in indicated CD4+ T cells polarized under Treg conditions for 48 hours (n ≥ 4 per genotype per group). (I) Immunoblot analysis of Anapc13 protein in CD4+ T cells polarized under Treg conditions shown in (H). The number on the top of the blot is the relative mean intensity of each Anapc13 band, and the right panel is the summary of the relative mean intensity. *P < 0.05 and **P < 0.01 (two-tailed Student’s t test).

Sf3b1-K700E–induced aberrant splicing event down-regulates Anapc13 gene critical for Treg differentiation

To determine whether lower levels of Anapc13 are responsible for the impaired Treg differentiation observed in Sf3b1K700Efl/+/CD4Cre CD4+ T cells, the effects of retrovirus-mediated expression of Anapc13 on Treg differentiation were determined. CD4+ T cells were labeled with dye for monitoring the proliferation of CellTrace Violet (CTV; fig. S6A). Forced expression of Anapc13 stimulated Treg differentiation (Fig. 6A) and cell proliferation (Fig. 6B) in Sf3b1K700Efl/+/CD4Cre but not Sf3b1K700Efl/+ CD4+ T cells (fig. S6B). Similar stimulation of Treg differentiation (Fig. 6C) and proliferation (Fig. 6D) by forced expression of Anapc13 was also observed in Sf3b1K700Efl/+/Foxp3YFP-Cre cells, strongly supporting that the Sf3b1-K700E mutant decreases Anapc13 expression, which then impairs Treg differentiation by reducing cell proliferation.

Fig. 6. Sf3b1-K700E–induced aberrant splicing event down-regulates Anapc13 gene critical for Treg differentiation.

(A) Representative flow cytometric analysis (left) and the percentage (right) of Foxp3+CD4+ Treg among indicated genotypes of CD4+ T cells transduced with retrovirus expressing green fluorescent protein (GFP) ± Anapc13 and polarized for 48 hours under Treg conditions (n ≥ 4 per genotype per group). (B) Representative flow cytometric analysis (left) and the percentage (right) of the proliferative dye–labeled cells in the indicated peak of Sf3b1K700Efl/+/CD4Cre CD4+ T cells expressing GFP ± Anapc13 and polarized under Treg conditions shown in (A). (C) Representative flow cytometric analysis (left) and the percentage (right) of Foxp3+CD4+ Treg among Sf3b1K700Efl/+/Foxp3YFP-Cre CD4+ T cells transduced with retrovirus expressing GFP ± Anapc13 and polarized for 48 hours under Treg conditions (n ≥ 4 per genotype per group). (D) Representative flow cytometric analysis (left) and the percentage (right) of proliferative dye–labeled Sf3b1K700Efl/+/Foxp3YFP-Cre CD4+ T cells indicated peak, expressing GFP ± Anapc13 and polarized under Treg conditions shown in C (n ≥ 4 per genotype). (E) Schematic representation of the WT (top) and aberrantly spliced Anapc13 isoform. (F) Relative luciferase activity from the indicated reporter transfected into 293 T cells (n = 4 per genotype). EV, empty vector. Boxed region: cell population of interest. Data are from three experiments [(A) and (H); (B) to (E), right panels, presented as means ± SEM] or are from one representative of three independent experiments [(B) to (E), left panels]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0005 (two-tailed Student’s t test).

To determine how the Sf3b1-K700E–induced aberrant splicing event affects Anapc13 gene expression, we designed primers to monitor the alternative splicing event based on our RNA-seq data. There were no alternative splicing events detected in naive CD4+ T cells from Sf3b1K700Efl/+/CD4Cre and Sf3b1K700Efl/+ mice (fig. S6D, top). However, Treg differentiation induced the alternative splicing event, indicated by the appearance of a high–molecular weight band, in Sf3b1K700Efl/+/CD4Cre cells expressing the Sf3b1-K700E mutant but not Sf3b1K700Efl/+ cells that does not express the Sf3b1-K700E mutant (fig. S6D, middle). The alternative splicing event was not detected in purified Tregs from spleens before or after stimulation (fig. S6E), which correlates with no proliferation defects of stimulated purified Tregs (fig. S1I). We then performed sequence analysis of the WT and alternatively spliced bands and confirmed that alternative usage of 5SS results in an insertion of a 231–base pair (bp) fragment between exon 1 and exon 2 (fig. S6C). Since the start codon ATG is located in exon 2 (Fig. 6E), exon 1 and the inserted 231-bp fragment are in the 5′UTR. To determine whether the inserted fragment affects gene expression, a WT DNA fragment containing exon 1 and exon 2 with or without alternative spliced 231-bp DNA fragment (Fig. 6E) was cloned between the SV40 promoter and a luciferase reporter gene. Insertion of 231 bp substantially inhibited luciferase activity (Fig. 6F). Deletion of a portion (about 80 bp) from the 231-bp insertion reduced inhibitory effects on luciferase activity (fig. S6F). Therefore, the Sf3b1-K700E–induced aberrant splicing event leads to the insertion of the 231-bp fragment that inhibits the expression of Anapc13 critical for Treg differentiation via controlling cell proliferation.

Forced expression of Anapc13 restores the function of Sf3b1K700Efl/+/CD4Cre Tregs in the prevention of colitis

To determine whether reduced Anapc13 is responsible for the impaired function of Sf3b1K700Efl/+/CD4Cre Tregs in the prevention of colitis, an adoptive transfer colitis model was used to determine the function of Anapc13 in the impaired function of Sf3b1K700Efl/+/CD4Cre Tregs. Naive CD4+ T cells from Sf3b1K700Efl/+/CD4Cre mice were transduced with retrovirus expressing green fluorescent protein (GFP; +EV) along or together with Anapc13 (+Anapc13) and differentiated into Tregs, followed by adoptive transfer of Treg differentiated GFP+ cells to Rag1−/− mice to inhibit colitis induced by adoptively transferred naive CD4+ T cells (fig. S7A). Overexpression of Anapc13 is confirmed 48 hours after viral infection (fig. S7B). Although severe colitis was induced by adoptive transfer of naive CD4+ along, the colitis was greatly inhibited by cotransfer of Sf3b1K700Efl/+/CD4Cre Tregs expressing Anapc13 but not GFP alone, indicated by the prevention of weight loss (Fig. 7A), shortening colon (Fig. 7B), tissue damages (Fig. 7C), and reduced IFN-γ but not IL-17 production from CD4+ T cells [see Fig. 7D and fig. S7 (C and D) for gating strategy] together with increased Tregs detected in colon and lymph nodes [see Fig. 7E and fig. S7 (C and D) for gating strategy]. Therefore, Sf3b1-K700E impairs Treg function via induction of an aberrant splicing event to inhibit Anapc13 expression.

Fig. 7. Forced expression of Anapc13 rescues Treg differentiation and function.

(A) Body weight of Rag1−/− recipients over time after adoptive transfer of WT CD45RBhiCD25−CD4+ naive T cells alone or in combination with differentiated iTregs retrovirally expressing GFP (EV) along or together with Anapc13 (+Anapc13). (B) Representative image of colons (left) and colon length (right) of colitis-induced recipient shown in (A) (n ≥ 4 per genotype). (C) H&E-stained colon sections from colitis-induced recipients shown in (A). (D) Representative flow cytometric analysis (left) and percentage (right) of CD4+IL-17A+ and CD4+IFN-γ+ cells recovered from colons of colitis-induced recipients shown in (A) (n ≥ 4 per group). (E) Representative flow cytometric analysis (left) and percentage (right) of Foxp3+CD4+ Tregs recovered from colon, spleen, and mLN of colitis-induced recipients shown in (A) (n ≥ 4 per group). Boxed area: cell population of interest. Data are from three experiments [(A); (B), (D), and (E), right panels, presented as means ± SEM] or are from one representative of three independent experiments [(C); (B), (D), and (E), left panels]. *P < 0.05 and **P < 0.01 (two-tailed Student’s t test).

AML grows faster in aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice

To determine whether Treg-specific expression of Sf3b1-K700E affects cancer development, we compared the growth of AML (acute myeloid leukemia) in Foxp3YFP-Cre and Sf3b1K700Efl/+/Foxp3YFP-Cre mice (fig. S8A). Sf3b1-K700E somatic mutation in AML was reported (45) and also identified by public cohort mining of DNA sequence from The Cancer Genome Atlas program (fig. S8B). We first noticed that aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice had much larger spleen size and weight than aged WT Foxp3YFP-Cre control mice (Fig. 8A). On the other hand, young Sf3b1K700Efl/+/Foxp3YFP-Cre mice had smaller spleen size and weight compared to young WT Foxp3YFP-Cre control mice. Transplanted AML cells (MLL-AF9-GFP) grew faster in aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice compared to aged Foxp3YFP-Cre control mice (Fig. 8B). In contrast, AML grew rather slower in young Sf3b1K700Efl/+/Foxp3YFP-Cre mice compared to young Foxp3YFP-Cre control mice. Consistently, the survival curves showed that more aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice died of AML compared to aged Foxp3YFP-Cre mice, whereas younger Sf3b1K700Efl/+/Foxp3YFP-Cre mice survived better compared to young Foxp3YFP-Cre mice (Fig. 8C). Analysis of GFP+ AML cells in peripheral blood (PB; see Fig. 8D and fig. S8C for gating strategy) and bone marrow (BM; see Fig. 8E and fig. S8D for gating strategy) confirmed that aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice had higher percentage of AML cells compared to aged Foxp3YFP-Cre mice. On the other hand, young Sf3b1K700Efl/+/Foxp3YFP-Cre mice had a lower percentage of AML cells compared to young Foxp3YFP-Cre mice. Therefore, our results demonstrate that SF3B1-K700E mutation in Tregs promotes AML growth in aged but not young mice. The faster growth of AML in aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice correlates with observed more inflammation (Fig. 4), whereas the slower AML growth in young Sf3b1K700Efl/+/Foxp3YFP-Cre mice correlates with less inflammation (Fig. 4), indicating the link between AML growth and inflammation.

Fig. 8. AML grows faster in aged Sf3b1K700Efl/+/Foxp3YFP-Cre mice.

(A) Representative image (left) and weight (right) of the spleens from indicated mice implanted with AML. (B) The percentage of GFP+ AML cells in peripheral blood (PB) of indicated mice at different times after implantation of AML (n = 7 per genotype per group). (C) Kaplan-Meier curves showing the survival of the indicated mice implanted with AML (n = 7 per genotype per group). (D and E) Representative flow cytometric analysis (left) and percentage (right) of GFP+ AML cells in the PB (E) and bone marrow [BM, (F)] from indicated mice 21 days after AML implantation. Boxed area: cell population of interest. Data are from three experiments [(A) and (B); (C) to (E), right panels, presented as means ± SEM] or are from one representative of three independent experiments [(C) to (E), left panels]. The statistical significance for (B) was determined using the log-rank (Mantel-Cox) test. *P < 0.05 and **P < 0.01 (two-tailed Student’s t test).

DISCUSSION

Hotspot heterozygous point mutations in SF3B1 are the most common across cancer types including CLL (46, 47), MDS (4, 13), and uveal melanoma (48–50). Previous research focuses on understanding how SF3B1 mutations, particularly the highly frequent SF3B1-K700E mutation, promote tumorigenesis (14–16, 51). SF3B1 mutations are identified in hematopoietic progenitors that differentiate into immune cells including Tregs. Here, we demonstrated that Treg-specific expression of SF3B1-K700E broke immune tolerance, resulting in autoinflammation. Mechanically, we showed that SF3B1-K700E induced an aberrant splicing event at the Anapc13 gene, which led to an insertion of a 231-bp fragment to the 5′UTR region. This insertion inhibited the expression of Anapc13 which is required for the proliferation of Tregs.

Anapc13 is a critical subunit of anaphase-promoting complexes (APC) which regulates cell cycle progression by controlling the degradation of cell cycle regulators including securing and cyclin B (52). APC has ubiquitin ligase activity and thus regulates the degradation of cell cycle regulators via ubiquitination-dependent pathways. Sequential degradation of different cell cycle regulators is a critical mechanism for controlling cell cycle progression. Inhibition of APC-dependent degradation prevents cell cycle progression. Anapc13 is a less studied protein compared to the other subunits of APC complexes. Anapc13 is important for stabilizing the APC complexes and is required for the ubiquitin ligase activity of APC. Deletion of Anapc13 stalls cell cycle progression (44). Consistent with the critical function of Anapc13 in the regulation of cell cycle progression, Sf3b1K700Efl/+/CD4Cre Tregs that express lower levels of Anapc13 display reduced proliferation. Reduced proliferation is responsible for impaired Sf3b1K700Efl/+/CD4Cre Treg differentiation, as forced expression of Anapc13 restores proliferation and Treg differentiation. Furthermore, forced expression of Anapc13 in Sf3b1K700Efl/+/CD4Cre Tregs restores its function in preventing colitis in vivo. Therefore, we demonstrated that SF3B1-K700E regulates Treg differentiation via regulating Anapc13-dependent cell cycle progression. SF3B1-K700E has been shown to regulate tumor cell proliferation via other mechanisms (16, 53). Therefore, how SF3B1 mutants affect cellular function is cell type–dependent, which is likely due to differential alternative splicing events induced by SF3B1-K700E in different cell types. Our study shows that SF3B1 induces a 3′ alternative splicing event at the Anapc13 gene in Tregs, which results in an insertion of a 231-bp fragment at 5′UTR. Furthermore, we show that insertion of the 231-bp fragment inhibits Anapc13 transcription. Insertion of the 231-bp fragment in front of the luciferase gene also inhibits luciferase activity, suggesting that this inhibition mechanism may not be gene-specific. It is thus worth investigating how the insertion of this fragment inhibits gene expression in the future.

Tregs are a double-edged sword for tumor development. On one hand, the tumor microenvironment, particularly solid tumors, is enriched with immunosuppressive cells including Tregs that allow tumor cells to escape from immune surveillance and prevent antitumor immune responses (54). On the other hand, inflammation resulting from impaired Treg differentiation and function has been shown to fuel tumor progression and metastasis (55–58). We showed that Treg-specific expression of SF3B1-K700E generates an inflammatory environment, indicated by pro-inflammatory cytokine production and lymphocyte infiltration. It remains unknown about the relationship between our observed SF3B1-K700E–induced inflammation and tumor development. Large-scale cancer genome sequencing projects have identified recurrent somatic mutations in splicing factors in several types of hematological malignancies including MDS and AML (4–6). Patients with MDS and AML have been found to produce a variety of inflammatory cytokines (59–63), and these inflammatory mediators have been shown to directly support the growth of stem cells containing SF3B1 mutation, resulting in the development of animal models of MDS (59, 64–67). Our results show that AML grows faster in aged, but not young, mice expressing SF3B1-K700E in Tregs. The increased AML growth is associated with heightened inflammation observed in aged mice. Cancer and inflammation also dominate in aged humans (59, 68–70), which shows the link between cancer and inflammation. However, it remains to be determined how the inflammation resulting from SF3B1-K700E–impaired Treg function promotes AML development.

Splicing factor mutations have been associated with different types of cancers including both hematological malignancies and solid tumors (71). However, so far, most studies focus on revealing how splicing factor mutations intrinsically promote oncogenesis. Little is known about how the splicing factor mutations affect the function of other somatic tissues. Our study demonstrates that splicing factor mutations could substantially interfere with other cell functions, immune cells in this study in addition to tumor cells. Furthermore, splicing factor mutations that affect the function of other tissues could contribute to tumor development. Understanding these extrinsic effects of splicing factor mutations will facilitate the development of effective therapies for the treatment of splicing factor mutation-associated cancers in general.

MATERIALS AND METHODS

Mice

Transgenic CD4Cre (TgCd4cre, 022071), Rag1−/− (Rag1tm1Mom, 002216), and C57BL (B6, 000664) mice were purchased from the Jackson Laboratory. Sf3b1K700E/fl mice were obtained from L.W. Laboratory (Systems Biology-BRI, Beckman Research Institute, City of Hope, CA). OT-II mice were obtained from J. Yu Laboratory (Department of Hematology and Hematopoietic Cell Transplantation, City of Hope, CA), and Foxp3YFP-Cre mice were obtained from M. Boldin Laboratory (Molecular and Cellular Biology, Beckman Research Institute, City of Hope, CA). All mice were bred into the C57BL/6J background and housed under specific pathogen–free conditions in the Animal Resource Center at the Beckman Research Institute of City of Hope under protocols approved by the Institutional Animal Care and Use Committee (IACUC; #07023). Mice were 10 to 12 weeks of age for EAE studies and 8 to 10 weeks of age for other experiments, unless indicated otherwise, with littermates age- and sex-matched across experimental groups.

Antibodies and cytokines

Monoclonal antibodies against mouse CD3 (145-2C11), CD28 (37.51), IL-4 (11B11), and IFN-γ (XMG1.2), as well as phycoerythrin (PE)-conjugated anti-CD8 (dilution ratio, 1:100; 53-6.7), allophycocyanin-conjugated anti-Foxp3 (dilution ratio, 1:100; FJK-16s), PE-indotricarbocyanine (Cy7)–conjugated anti–IL-17A (dilution ratio, 1:100; eBio17B7), APC-conjugated anti–IFN-γ (dilution ratio, 1:100; XMG1.2), and Live/Dead Fixable Near-IR Dead Cell Stain (dilution ratio, 1:1000; L34976) were from Invitrogen. PE-conjugated anti-CD25 (dilution ratio, 1:100; PC61), Brilliant Violet (BV) 605–conjugated anti-CD4 (dilution ratio, 1:100; RM4-5), BV 421–conjugated anti-CD3 (dilution ratio, 1:100; 145-2C11), APC-conjugated anti-CD45 (dilution ratio, 1:100; I3/2.3), PE-Cy7–conjugated anti-CD45RB (dilution ratio, 1:100; C363-16A), PE-Cy7–conjugated anti-CD62L (dilution ratio, 1:100; MEL-14), APC-Cy7–conjugated anti-CD44 (dilution ratio, 1:100; IM7), PE-conjugated anti-CD73 (dilution ratio, 1:100; TY/11.8), PE-Cy7–conjugated anti-CD39 (dilution ratio, 1:100; Duha59), APC-Cy7–conjugated anti-CD45 (dilution ratio, 1:100; 30-F11), PE-Cy7–conjugated anti–CTLA-4 (dilution ratio, 1:100; UC10-4B9), and recombinant murine IL-2 were from BioLegend. APC-conjugated anti-CD25 (dilution ratio, 1:100; PC61) was from BD Biosciences. Antibodies against Anapc13 (dilution ratio, 1:1000; PA5-20956, Thermo Fisher Scientific) and β-actin (dilution ratio, 1:1000; 4970L, Cell Signaling) were used for immunoblotting analysis. Recombinant mouse TGF-β was from Miltenyi Biotec.

Plasmids

The retroviral vector murine stem cell virus (MSCV)–internal ribosomal entry site (IRES)–GFP was a gift from W. S. Pear (University of Pennsylvania). cDNA encoding Anapc13 and ATF3 was cloned into MSCV-IRES-GFP vector. IRF4-MIEG-GFP was a gift from M. H. Kaplan Laboratory (Indiana University School of Medicine); 7×E-Box:Renilla (plasmid #124532) and MSCV-PIG-Myb (plasmid #66988) were purchased from Addgene. pGL3-promoter (plasmid #E1761) was purchased from Promega (Madison, WI).

Flow cytometry

For surface staining, cells isolated from mice or in vitro culture were directly stained with antibodies and/or fixable live/dead dye with 2% fetal bovine serum (FBS) and 1 mM EDTA at 4°C for 15 min. For transcription factor staining, cells prestained with surface markers were fixed and permeabilized in TF Fix/Perm buffer (BD Biosciences) at 4°C for 20 min, washed once with TF Perm/Wash buffer, and stained with target markers in the TF Perm/Wash buffer at 4°C for 15 min. For intracellular cytokine analysis, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma-Aldrich) and ionomycin (750 ng/ml; Sigma-Aldrich) at 37°C for 3 hours in the presence of GolgiStop (BD Biosciences) before staining. After stimulation, cells were stained with surface markers and then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) for 20 min followed by staining cytokines in the Perm/Wash buffer (BD Biosciences) after washing. The expression of surface and intracellular markers was analyzed with a BD LSRFortessa flow cytometer.

Isolation of naive CD4+ T cells and in vitro Treg differentiation

Naive CD4+ T cells were isolated from mouse spleens by negative selection using the Naive CD4+ T Cell Isolation Kit (Miltenyi Biotec). Suspensions of 3 × 105 cells per well of RPMI 1640 medium (Corning Inc.) containing 2 mM l-glutamine, 50 μM β-mercaptoethanol, penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% FBS (Corning Inc.) were cultured in 48-well plates precoated with rabbit anti-hamster (0.1 mg/ml). The medium was supplemented with hamster anti-CD3 (0.25 μg/ml), hamster anti-CD28 (1 μg/ml), TGF-β (5 ng/ml), anti–IL-4 (2.5 μg/ml), and anti–IFN-γ (2.5 μg/ml) for Treg differentiation for up to 48 hours.

In vivo induction of iTregs by adoptively transferring naive CD4+ T cells

Splenic cells were collected from Foxp3YFP-Cre or Sf3b1K700E/fl/Foxp3YFP-Cre mice (8 to 10 weeks). Naive CD4+ T cells were first enriched by negative selection using the Naive CD4+ T Cell Isolation Kit, and then CD4+YFP− cells were sorted via FACSAria Fusion (BD Biosciences) to enable a high purity of ≥99.0%. A total of 4 × 105 naive CD4+ T cells were intraperitoneally injected into sex-matched Rag1−/− mice. Three weeks after adoptive transfer, cells from the spleen and mLN of Rag1−/−-recipient mice were collected and analyzed.

In vivo induction of iTregs by oral tolerance

Splenic cells were collected from OT-II/ Sf3b1K700E/fl or OT-II/ Sf3b1K700E/fl /CD4Cre mice (8 to 10 weeks), naive CD4+ T cells were first enriched by negative selection using the Naive CD4+ T Cell Isolation Kit, and CD4+CD25− cells were then sorted via FACSAria Fusion to enable a high purity of ≥99.0%. A total of 3 × 106 cells were intraperitoneally injected into sex-matched Rag1−/− mice. After 24 hours, recipient mice were provided with grade VI OVA (20 mg/ml; Sigma-Aldrich) ad libitum in drinking water for 5 days. Drinking water containing OVA was changed every 2 days. Cells were collected from the colon, spleen, and mLN on day 6 for analysis.

Induction and assessment of EAE

EAE was induced and assessed according to the manufacturer’s instructions (Hooke Laboratories, Lawrence, MA). Briefly, Foxp3YFP-Cre or Sf3b1K700E/fl /Foxp3YFP-Cre mice were immunized with 200 mg of MOG35-55 (Hooke Laboratories) in complete Freund’s adjuvant by subcutaneous injection at two dorsal sites of mice, followed by two intraperitoneal injections of 80 ng of pertussis toxin at days 0 and 1. The severity of EAE was monitored and evaluated on a scale from 0 to 5 according to Hooke Laboratories’ guidelines. Briefly, 0 represents no disease, 1 represents a paralyzed tail, 2 represents hindlimb weakness, 3 represents hindlimb paralysis, 4 represents hindlimb and forelimb paralysis, and 5 represents moribund and death. When a mouse was euthanized because of severe paralysis, a score of 5 was entered for that mouse for the rest of the experiment.

In vivo Treg suppression assay

Colitis was induced in sex-matched Rag1−/− mice by intraperitoneally injecting 4 × 105 CD45RBhiCD25−CD4+ naive T cells sorted from the spleen of C57BL mice (8 to 10 weeks). For nTreg suppression assay in Sf3b1K700E/fl /Foxp3YFP-Cre strain mice, 2 × 105 CD4+YFP+ Tregs sorted from the spleen of 8- to10-week-old Foxp3YFP-Cre or Sf3b1K700E/fl /Foxp3YFP-Cre mice were mixed with 4 × 105 CD45RBhiCD25−CD4+ naive T cells from C57BL mice and injected into sex-matched Rag1−/− mice.). For nTreg suppression assay in Sf3b1K700E/fl /CD4Cre strain mice, 2 × 105 CD4+CD25+ Tregs sorted from the spleen of 8- to 10-week-old Sf3b1K700E/fl or Sf3b1K700E/fl/CD4Cre mice were mixed with 4 × 105 CD45RBhiCD25−CD4+ naive T cells from C57BL mice and injected into sex-matched Rag1−/− mice. For the iTreg suppression assay, naive CD4+ T cells from Sf3b1K700E/fl /CD4Cre mice were in vitro activated as above and retrovirally transducted with EV or Anapc13 alone. After differentiation for 3 days, 2 × 105 iTregs transducted with EV or Anapc13 alone were mixed with 4 × 105 CD45RBhiCD25−CD4+ naive T cells from C57BL mice and injected into sex-matched Rag1−/− mice as above. Mice were weighed immediately following T cell transfer and weekly thereafter. Eight to 9 weeks after cell transfer, the colon, spleen, and mLN were removed from Rag1−/−-recipient mice for analysis.

In vitro Treg suppression assay

Sorted CD4+CD25− T cells were labeled with CTV (dilution ratio, 1:1000; C34557, Invitrogen) and served as responder CD4+ T (Tresp) cells. Tresp cells (6 × 105 cells/ml) were cocultured with CD4+YFP+ Tregs sorted from the spleens of Foxp3YFP-Cre or Sf3b1K700E/fl /Foxp3YFP-Cre mice in 48-well plates [precoated with rabbit anti-hamster (0.1 mg/ml)] in culture medium supplemented with hamster anti-CD3 (0.25 μg/ml) and hamster anti-CD28 (1 μg/ml) for 3 days. The ratios of Tresp cells to Tregs were 1:0, 1:1, and 2:1 for Tregs sorted from mice and 1:0, 1:1, and 2:1 for Tregs sorted from in vitro differentiation. The proliferation of Tresp cells was assessed by flow cytometry.

Histology study

Tissues were cleaned and fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned and stained with hematoxylin and eosin.

RNA-seq and analysis

Naive CD4+ T cells isolated from Sf3b1K700E/fl or Sf3b1K700E/fl /Cd4Cre mice were differentiated into Tregs in 24-well plates in the presence of TGF-β (5 ng/ml), anti–IL-4, and anti–IFN-γ for 36 hours. CD4+ T cells after 36 hours of Treg differentiation were collected and subjected to RNA extraction with the RNeasy Mini Kit (QIAGEN). Each group has three replicates from different mice. Quality control, library preparation, and sequencing were performed at Novogene. The analysis was performed through Partek Flow. Briefly, the sequence reads were aligned to the mouse whole genome (GRCm38) with validation of quality through pre-alignment and post-alignment quality assurance/quality control. Aligned reads were further subjected to quantification using the Partek E/M algorithm and normalization to counts per million with 0.001 added to each. The identification of differentially expressed features was performed through the Partek GSA algorithm that applies multiple statistical models to each gene. Genes with total counts of more than 10 were considered to be statistically expressed in the cells. The expression values of pathogenic genes were extracted and subjected to ingenuity pathway analysis and network analysis.

Differential alternative splicing analyses

Alternative splicing analysis was performed using rMATS (version 4.0.2) (72), a Python algorithm used to identify alternative splicing events by quantifying exon-exon junction spanning reads on annotated splice junctions in rat GENCODE Rnor_6.0 assembly. Differentially spliced mRNAs were defined as a false discovery rate of <0.05 and a minimum inclusion level difference of >10% or <−10%. Three mutant Sf3b1K700E replicates and three WT replicates were compared.

Reverse transcription quantitative real-time PCR

Total RNA of cells was extracted according to the manufacturer’s guidelines using the RNeasy Mini Kit (QIAGEN). The first-strand cDNA synthesis was performed by reverse transcription using a Tetro cDNA Synthesis Kit (Bioline). Subsequent qPCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) in the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The primers used for qPCR and reverse transcription PCR are listed in table S1. The amplification efficiency of all primers has been tested, and the optimized conditions were used in all qPCRs. Gene expression was calculated with the ΔΔCt method normalized to the control gene encoding β-actin, and all measurements were performed in triplicate.

Retroviral transduction

Vectors were firstly transfected to Platinum-E (Plat-E; Cell Biolabs) retroviral packaging cells by using BioT transfection reagent (Bioland Scientific) followed by a changing fresh medium at 24 hours. The virus-containing medium collected at 48 and 72 hours was filtered with a 0.45-μm polyvinylidene difluoride (PVDF) syringe filter (Millipore), followed by either direct transduction to T cells or stored at −80°C for later use. Naive CD4+ T cells were labeled with Cell Trace Violet (dilution ratio, 1:1000; C34557, Invitrogen) and then activated by hamster anti-CD3 (0.25 μg/ml) and hamster anti-CD28 antibodies (1 μg/ml) in precoated plates for 20 hours before transduction. Transduction to activated CD4+ T cells was performed by spin infection with viral supernatants (2500 g, 30°C for 2 hours) in the presence of polybrene (10 μg/ml; Sigma-Aldrich). Afterward, the plates were kept in the incubator at 37°C for 3 hours. The viral supernatant was replaced by a fresh culture medium with polarizing cytokines and antibodies for Treg differentiation.

Western blotting

For Western blotting, cells were lysed in radioimmunoprecipitation assay buffer containing 20 mM tris-HCl (pH 7.4), 150 mM NaCl,1 mM Na2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and leupeptin (1 μg/ml) on ice for 45 min and spun down at 15,000 rpm for 10 min at 4°C to collect the extract. The 2× Laemmli sample buffer (Bio-Rad) containing β-mercaptoethanol was mixed with cell extract and heated at 95°C for 5 min. Protein was separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore). Target proteins were sequentially immunoblotted with relevant primary antibodies and fluorescent secondary antibodies (LI-COR Biosciences) followed by measuring fluorescent intensity with LI-COR Odyssey blot imager (LI-COR Biosciences).

Luciferase assay

The 5′UTR DNA sequence of Anapc13 as the WT sequence and the 5′UTR DNA sequence including the insertion of the 231-bp DNA sequence of Anapc13 as the Mut sequence. Both of the sequences were inserted between the promoter and luciferase of the basic PGL3 promoter vector. To measure the luciferase activity, the PGL3-promoter vector (1 μg), PGL3-promoter-WT, or PGL3-promoter-Mut luciferase vector alone (1 μg) was delivered to 4 × 105 human embryonic kidney 293T cells seeded in a six-well plate. Renilla luciferase vector (200 ng) was cotransfected to cells in each group for normalizing different transfection efficiencies. An empty vector was used to adjust the total plasmid DNA to the same amount. Luciferase activity was measured in Dual-Luciferase Reporter Assay System (Promega, Madison, WI) per the manufacturer’s instruction at 1-day posttransfection in a Synergy HTX multi-mode reader (Agilent, Santa Clara, CA). Briefly, after background subtraction, Firefly luciferase activities were normalized to Renilla luciferase values, followed by an additional normalization of all Firefly to Renilla ratios to the PGL3-promoter group.

AML mouse model

Murine MLL-AF9-GFP cell (C58BL/6) was a gift from Y.H. Kuo (City of Hope). MLL-AF9-GFP AML cells were transplanted (1 × 106 cells) via tail vein intravenous (iv) injection into recipient mice to generate AML. Engraftment of GFP+ cells in PB was monitored by flow cytometry every week after transplantation. After 3 weeks, PB and BM cells were collected and assessed for AML burden by flow cytometry.

Statistics and reproducibility

The results were analyzed for statistical significance with unpaired Student’s t test or one-way or two-way analysis of variance (ANOVA) where appropriate. The log-rank test was used to assess significant differences between survival curves. All data are presented as means ± SEM. P values are calculated using GraphPad Prism and presented where the statistical significance (P < 0.05) was found.

Acknowledgments

We thank W. S. Pear and M. H. Kaplan for sharing the plasmids of MSCV-IRES-GFP and IRF4-MIEG-GFP, respectively. We thank Y. H. Kuo for sharing the MLL-AF9-GFP cell. We also thank L.W., J. Yu, and M. Boldin for sharing the mice strain of Sf3b1K700E/fl, OT-II, and Foxp3YFP-Cre, respectively. Besides, we appreciate the help from the City of Hope core facilities: Animal Resource Center, Integrative Genomics Core, Pathology Solid Tumor Core, and Bioinformatics Core. We thank Dr. C. S. Jayasena (City of Hope, Duarte, CA) for helpful comments and for editing the manuscript. We also would like to acknowledge the CoH Center for Informatics for using the POSEIDON platforms for data exploration, visualization, analysis, and discovery.

Funding: This work was supported by grants from NIH R01-AI109644, R21-AI163256, institutional pilot funding, Jackie and Bruce Barrow Cancer Research Scholars’Program, AR-DMRI Innovative grant, and Caltech-CoH Biomedical Initiative. Research reported in this publication included work performed in the animal, genomic, and flow cytometry cores supported under NIH grant P30CA033572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author contributions: Conceptualization: Z.S. and Y.S. Methodology: Y.S., W.Z., X.Z., H.W., L.Z., P.I., Y.-C.Y., and Y.Z. Investigation: Y.S., W.Z., X.Z., and H.W. Visualization: Y.S., W.Z., X.Z., Q.J., and H.W. Supervision: Z.S. Writing—original draft: Z.S. and Y.S. Writing—review and editing: Z.S., Y.S., L.W., Z.J., and Y.-H.K.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The high-throughput sequence data can be accessed at GEO via record #GSE201431.

Supplementary Materials

This PDF file includes:

Figs. S1 to S8

Table S1
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REFERENCES AND NOTES

1 M. Cazzola, M. Rossi, L. Malcovati, M. Associazione, Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie, biologic and clinical significance of somatic mutations of SF3B1 in myeloid and lymphoid neoplasms. Blood 121 , 260–269 (2013).23160465
2 M. Chen, J. L. Manley, Mechanisms of alternative splicing regulation: Insights from molecular and genomics approaches. Nat. Rev. Mol. Cell Biol. 10 , 741–754 (2009).19773805
3 W. Chen, M. J. Moore, The spliceosome: Disorder and dynamics defined. Curr. Opin. Struct. Biol. 24 , 141–149 (2014).24530854
4 K. Yoshida, M. Sanada, Y. Shiraishi, D. Nowak, Y. Nagata, R. Yamamoto, Y. Sato, A. Sato-Otsubo, A. Kon, M. Nagasaki, G. Chalkidis, Y. Suzuki, M. Shiosaka, R. Kawahata, T. Yamaguchi, M. Otsu, N. Obara, M. Sakata-Yanagimoto, K. Ishiyama, H. Mori, F. Nolte, W. K. Hofmann, S. Miyawaki, S. Sugano, C. Haferlach, H. P. Koeffler, L. Y. Shih, T. Haferlach, S. Chiba, H. Nakauchi, S. Miyano, S. Ogawa, Frequent pathway mutations of splicing machinery in myelodysplasia. Nature 478 , 64–69 (2011).21909114
5 L. Malcovati, K. Stevenson, E. Papaemmanuil, D. Neuberg, R. Bejar, J. Boultwood, D. T. Bowen, P. J. Campbell, B. L. Ebert, P. Fenaux, T. Haferlach, M. Heuser, J. H. Jansen, R. S. Komrokji, J. P. Maciejewski, M. J. Walter, M. Fontenay, G. Garcia-Manero, T. A. Graubert, A. Karsan, M. Meggendorfer, A. Pellagatti, D. A. Sallman, M. R. Savona, M. A. Sekeres, D. P. Steensma, S. Tauro, F. Thol, P. Vyas, A. A. Van de Loosdrecht, D. Haase, H. Tuchler, P. L. Greenberg, S. Ogawa, E. Hellstrom-Lindberg, M. Cazzola, SF3B1-mutant MDS as a distinct disease subtype: A proposal from the International Working Group for the Prognosis of MDS. Blood 136 , 157–170 (2020).32347921
6 E. Papaemmanuil, M. Gerstung, L. Malcovati, S. Tauro, G. Gundem, P. Van Loo, C. J. Yoon, P. Ellis, D. C. Wedge, A. Pellagatti, A. Shlien, M. J. Groves, S. A. Forbes, K. Raine, J. Hinton, L. J. Mudie, S. McLaren, C. Hardy, C. Latimer, M. G. D. Porta, S. O’Meara, I. Ambaglio, A. Galli, A. P. Butler, G. Walldin, J. W. Teague, L. Quek, A. Sternberg, C. Gambacorti-Passerini, N. C. P. Cross, A. R. Green, J. Boultwood, P. Vyas, E. Hellstrom-Lindberg, D. Bowen, M. Cazzola, M. R. Stratton, P. J. Campbell; Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium, Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood 122 , 3616–3627 (2013).24030381
7 G. Garcia-Manero, Myelodysplastic syndromes: 2012 update on diagnosis, risk-stratification, and management. Am. J. Hematol. 87 , 692–701 (2012).22696212
8 L. Malcovati, E. Papaemmanuil, D. T. Bowen, J. Boultwood, M. G. D. Porta, C. Pascutto, E. Travaglino, M. J. Groves, A. L. Godfrey, I. Ambaglio, A. Galli, M. C. Da Via, S. Conte, S. Tauro, N. Keenan, A. Hyslop, J. Hinton, L. J. Mudie, J. S. Wainscoat, P. A. Futreal, M. R. Stratton, P. J. Campbell, E. Hellstrom-Lindberg, M. Cazzola; Chronic Myeloid Disorders Working Group of the International Cancer Genome Consortium and of the Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative, Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms. Blood 118 , 6239–6246 (2011).21998214
9 L. Malcovati, M. Karimi, E. Papaemmanuil, I. Ambaglio, M. Jadersten, M. Jansson, C. Elena, A. Galli, G. Walldin, M. G. Della Porta, K. Raaschou-Jensen, E. Travaglino, K. Kallenbach, D. Pietra, V. Ljungstrom, S. Conte, E. Boveri, R. Invernizzi, R. Rosenquist, P. J. Campbell, M. Cazzola, E. Hellstrom Lindberg, SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Blood 126 , 233–241 (2015).25957392
10 T. Mortera-Blanco, M. Dimitriou, P. S. Woll, M. Karimi, E. Elvarsdottir, S. Conte, M. Tobiasson, M. Jansson, I. Douagi, M. Moarii, L. Saft, E. Papaemmanuil, S. E. W. Jacobsen, E. Hellstrom-Lindberg, SF3B1-initiating mutations in MDS-RSs target lymphomyeloid hematopoietic stem cells. Blood 130 , 881–890 (2017).28634182
11 Y. Shiozawa, L. Malcovati, A. Galli, A. Sato-Otsubo, K. Kataoka, Y. Sato, Y. Watatani, H. Suzuki, T. Yoshizato, K. Yoshida, M. Sanada, H. Makishima, Y. Shiraishi, K. Chiba, E. Hellstrom-Lindberg, S. Miyano, S. Ogawa, M. Cazzola, Aberrant splicing and defective mRNA production induced by somatic spliceosome mutations in myelodysplasia. Nat. Commun. 9 , 3649 (2018).30194306
12 P. Fenaux, U. Platzbecker, G. J. Mufti, G. Garcia-Manero, R. Buckstein, V. Santini, M. Diez-Campelo, C. Finelli, M. Cazzola, O. Ilhan, M. A. Sekeres, J. F. Falantes, B. Arrizabalaga, F. Salvi, V. Giai, P. Vyas, D. Bowen, D. Selleslag, A. E. DeZern, J. G. Jurcic, U. Germing, K. S. Gotze, B. Quesnel, O. Beyne-Rauzy, T. Cluzeau, M. T. Voso, D. Mazure, E. Vellenga, P. L. Greenberg, E. Hellstrom-Lindberg, A. M. Zeidan, L. Ades, A. Verma, M. R. Savona, A. Laadem, A. Benzohra, J. Zhang, A. Rampersad, D. R. Dunshee, P. G. Linde, M. L. Sherman, R. S. Komrokji, A. F. List, Luspatercept in patients with lower-risk myelodysplastic syndromes. N. Engl. J. Med. 382 , 140–151 (2020).31914241
13 E. Papaemmanuil, M. Cazzola, J. Boultwood, L. Malcovati, P. Vyas, D. Bowen, A. Pellagatti, J. S. Wainscoat, E. Hellstrom-Lindberg, C. Gambacorti-Passerini, A. L. Godfrey, I. Rapado, A. Cvejic, R. Rance, C. McGee, P. Ellis, L. J. Mudie, P. J. Stephens, S. McLaren, C. E. Massie, P. S. Tarpey, I. Varela, S. Nik-Zainal, H. R. Davies, A. Shlien, D. Jones, K. Raine, J. Hinton, A. P. Butler, J. W. Teague, E. J. Baxter, J. Score, A. Galli, M. G. D. Porta, E. Travaglino, M. Groves, S. Tauro, N. C. Munshi, K. C. Anderson, A. El-Naggar, A. Fischer, V. Mustonen, A. J. Warren, N. C. Cross, A. R. Green, P. A. Futreal, M. R. Stratton, P. J. Campbell; Chronic Myeloid Disorders Working Group of the International Cancer Genome, Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts. N. Engl. J. Med. 365 , 1384–1395 (2011).21995386
14 S. Yin, R. G. Gambe, J. Sun, A. Z. Martinez, Z. J. Cartun, F. F. D. Regis, Y. Wan, J. Fan, A. N. Brooks, S. E. M. Herman, E. Ten Hacken, A. Taylor-Weiner, L. Z. Rassenti, E. M. Ghia, T. J. Kipps, E. A. Obeng, C. L. Cibulskis, D. Neuberg, D. R. Campagna, M. D. Fleming, B. L. Ebert, A. Wiestner, I. Leshchiner, J. A. DeCaprio, G. Getz, R. Reed, R. D. Carrasco, C. J. Wu, L. Wang, A murine model of chronic lymphocytic leukemia based on b cell-restricted expression of Sf3b1 mutation and Atm deletion. Cancer Cell 35 , 283–296.e5 (2019).30712845
15 M. Cusan, H. Shen, B. Zhang, A. Liao, L. Yang, M. Jin, M. Fernandez, P. Iyer, Y. Wu, K. Hart, C. Gutierrez, S. Nik, S. M. Pruett-Miller, J. Stark, E. A. Obeng, T. V. Bowman, C. J. Wu, R. J. Lin, L. Wang, SF3B1 mutation and ATM deletion codrive leukemogenesis via centromeric R-loop dysregulation. J. Clin. Invest. 133 , e163325 (2023).37463047
16 Z. Liu, A. Yoshimi, J. Wang, H. Cho, S. Chun-Wei Lee, M. Ki, L. Bitner, T. Chu, H. Shah, B. Liu, A. R. Mato, P. Ruvolo, G. Fabbri, L. Pasqualucci, O. Abdel-Wahab, R. Rabadan, Mutations in the RNA splicing factor SF3B1 promote tumorigenesis through MYC stabilization. Cancer Discov. 10 , 806–821 (2020).32188705
17 G. Plitas, A. Y. Rudensky, Regulatory T cells: Differentiation and function. Cancer Immunol. Res. 4 , 721–725 (2016).27590281
18 C. L. Bennett, J. Christie, F. Ramsdell, M. E. Brunkow, P. J. Ferguson, L. Whitesell, T. E. Kelly, F. T. Saulsbury, P. F. Chance, H. D. Ochs, The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat. Genet. 27 , 20–21 (2001).11137993
19 M. E. Brunkow, E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, F. Ramsdell, Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27 , 68–73 (2001).11138001
20 R. S. Wildin, F. Ramsdell, J. Peake, F. Faravelli, J. L. Casanova, N. Buist, E. Levy-Lahad, M. Mazzella, O. Goulet, L. Perroni, F. D. Bricarelli, G. Byrne, M. McEuen, S. Proll, M. Appleby, M. E. Brunkow, X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27 , 18–20 (2001).11137992
21 M. A. Curotto de Lafaille, J. J. Lafaille, Natural and adaptive foxp3+ regulatory T cells: More of the same or a division of labor? Immunity 30 , 626–635 (2009).19464985
22 C. W. Lio, C. S. Hsieh, A two-step process for thymic regulatory T cell development. Immunity 28 , 100–111 (2008).18199417
23 M. A. Burchill, J. Yang, K. B. Vang, J. J. Moon, H. H. Chu, C. W. Lio, A. L. Vegoe, C. S. Hsieh, M. K. Jenkins, M. A. Farrar, Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire. Immunity 28 , 112–121 (2008).18199418
24 J. Ma, Y. Ding, X. Fang, R. Wang, Z. Sun, Protein kinase C-theta inhibits inducible regulatory T cell differentiation via an AKT-Foxo1/3a-dependent pathway. J. Immunol. 188 , 5337–5347 (2012).22539794
25 E. Bettelli, Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, V. K. Kuchroo, Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441 , 235–238 (2006).16648838
26 L. Zhou, J. E. Lopes, M. M. Chong, I. I. Ivanov, R. Min, G. D. Victora, Y. Shen, J. Du, Y. P. Rubtsov, A. Y. Rudensky, S. F. Ziegler, D. R. Littman, TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORγt function. Nature 453 , 236–240 (2008).18368049
27 E. A. Obeng, R. J. Chappell, M. Seiler, M. C. Chen, D. R. Campagna, P. J. Schmidt, R. K. Schneider, A. M. Lord, L. Wang, R. G. Gambe, M. E. McConkey, A. M. Ali, A. Raza, L. Yu, S. Buonamici, P. G. Smith, A. Mullally, C. J. Wu, M. D. Fleming, B. L. Ebert, Physiologic expression of Sf3b1(K700E) causes impaired erythropoiesis, aberrant splicing, and sensitivity to therapeutic spliceosome modulation. Cancer Cell 30 , 404–417 (2016).27622333
28 M. Yadav, C. Louvet, D. Davini, J. M. Gardner, M. Martinez-Llordella, S. Bailey-Bucktrout, B. A. Anthony, F. M. Sverdrup, R. Head, D. J. Kuster, P. Ruminski, D. Weiss, D. Von Schack, J. A. Bluestone, Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J. Exp. Med. 209 , 1713–1722 (2012).22966003
29 A. M. Thornton, P. E. Korty, D. Q. Tran, E. A. Wohlfert, P. E. Murray, Y. Belkaid, E. M. Shevach, Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J. Immunol. 184 , 3433–3441 (2010).20181882
30 W. Chen, Z. Xu, Y. Zheng, J. Wang, W. Qian, N. Olsen, D. Brand, J. Lin, S. G. Zheng, A protocol to develop T helper and Treg cells in vivo. Cell. Mol. Immunol. 14 , 1013–1016 (2017).29082917
31 R. Wang, S. Campbell, M. Amir, S. A. Mosure, M. A. Bassette, A. Eliason, M. S. Sundrud, T. M. Kamenecka, L. A. Solt, Genetic and pharmacological inhibition of the nuclear receptor RORα regulates TH17 driven inflammatory disorders. Nat. Commun. 12 , 76 (2021).33397953
32 E. M. Shevach, Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 30 , 636–645 (2009).19464986
33 A. Schmidt, N. Oberle, P. H. Krammer, Molecular mechanisms of Treg-mediated T cell suppression. Front. Immunol. 3 , 51 (2012).22566933
34 C. Konopacki, Y. Pritykin, Y. Rubtsov, C. S. Leslie, A. Y. Rudensky, Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function. Nat. Immunol. 20 , 232–242 (2019).30643266
35 L. F. Lu, M. P. Boldin, A. Chaudhry, L. L. Lin, K. D. Taganov, T. Hanada, A. Yoshimura, D. Baltimore, A. Y. Rudensky, Function of miR-146a in controlling Treg cell-mediated regulation of TH1 responses. Cell 142 , 914–929 (2010).20850013
36 W. Zhang, X. Cao, X. Zhong, H. Wu, M. Feng, Y. Gwack, N. Isakov, Z. Sun, Steroid nuclear receptor coactivator 2 controls immune tolerance by promoting induced Treg differentiation via up-regulating Nr4a2. Sci. Adv. 8 , eabn7662 (2022).35704583
37 S. Alsafadi, A. Houy, A. Battistella, T. Popova, M. Wassef, E. Henry, F. Tirode, A. Constantinou, S. Piperno-Neumann, S. Roman-Roman, M. Dutertre, M. H. Stern, Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat. Commun. 7 , 10615 (2016).26842708
38 C. Liu, H. C. Wang, S. Yu, R. Jin, H. Tang, Y. F. Liu, Q. Ge, X. H. Sun, Y. Zhang, Id1 expression promotes T regulatory cell differentiation by facilitating TCR costimulation. J. Immunol. 193 , 663–672 (2014).24920844
39 S. I. Koizumi, H. Ishikawa, Transcriptional regulation of differentiation and functions of effector T regulatory cells. Cells 8 , 939 (2019).31434282
40 G. Alvisi, J. Brummelman, S. Puccio, E. M. Mazza, E. P. Tomada, A. Losurdo, V. Zanon, C. Peano, F. S. Colombo, A. Scarpa, M. Alloisio, A. Vasanthakumar, R. Roychoudhuri, M. Kallikourdis, M. Pagani, E. Lopci, P. Novellis, J. Blume, A. Kallies, G. Veronesi, E. Lugli, IRF4 instructs effector Treg differentiation and immune suppression in human cancer. J. Clin. Invest. 130 , 3137–3150 (2020).32125291
41 R. B. Darman, M. Seiler, A. A. Agrawal, K. H. Lim, S. Peng, D. Aird, S. L. Bailey, E. B. Bhavsar, B. Chan, S. Colla, L. Corson, J. Feala, P. Fekkes, K. Ichikawa, G. F. Keaney, L. Lee, P. Kumar, K. Kunii, C. MacKenzie, M. Matijevic, Y. Mizui, K. Myint, E. S. Park, X. Puyang, A. Selvaraj, M. P. Thomas, J. Tsai, J. Y. Wang, M. Warmuth, H. Yang, P. Zhu, G. Garcia-Manero, R. R. Furman, L. Yu, P. G. Smith, S. Buonamici, Cancer-associated SF3B1 hotspot mutations induce Cryptic 3’ splice site selection through use of a different branch point. Cell Rep. 13 , 1033–1045 (2015).26565915
42 C. DeBoever, E. M. Ghia, P. J. Shepard, L. Rassenti, C. L. Barrett, K. Jepsen, C. H. Jamieson, D. Carson, T. J. Kipps, K. A. Frazer, Transcriptome sequencing reveals potential mechanism of cryptic 3’ splice site selection in SF3B1-mutated cancers. PLOS Comput. Biol. 11 , e1004105 (2015).25768983
43 L. Wang, A. N. Brooks, J. Fan, Y. Wan, R. Gambe, S. Li, S. Hergert, S. Yin, S. S. Freeman, J. Z. Levin, L. Fan, M. Seiler, S. Buonamici, P. G. Smith, K. F. Chau, C. L. Cibulskis, W. Zhang, L. Z. Rassenti, E. M. Ghia, T. J. Kipps, S. Fernandes, D. B. Bloch, D. Kotliar, D. A. Landau, S. A. Shukla, J. C. Aster, R. Reed, D. S. DeLuca, J. R. Brown, D. Neuberg, G. Getz, K. J. Livak, M. M. Meyerson, P. V. Kharchenko, C. J. Wu, Transcriptomic characterization of SF3B1 mutation reveals its pleiotropic effects in chronic Lymphocytic Leukemia. Cancer Cell 30 , 750–763 (2016).27818134
44 M. Schwickart, J. Havlis, B. Habermann, A. Bogdanova, A. Camasses, T. Oelschlaegel, A. Shevchenko, W. Zachariae, Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27. Mol. Cell. Biol. 24 , 3562–3576 (2004).15060174
45 D. Cilloni, F. Itri, V. Bonuomo, J. Petiti, SF3B1 mutations in hematological malignancies. Cancers 14 , 4927 (2022).36230848
46 V. Quesada, A. J. Ramsay, C. Lopez-Otin, Chronic lymphocytic leukemia with SF3B1 mutation. N. Engl. J. Med. 366 , 2530 (2012).22738114
47 L. Wang, M. S. Lawrence, Y. Wan, P. Stojanov, C. Sougnez, K. Stevenson, L. Werner, A. Sivachenko, D. S. DeLuca, L. Zhang, W. Zhang, A. R. Vartanov, S. M. Fernandes, N. R. Goldstein, E. G. Folco, K. Cibulskis, B. Tesar, Q. L. Sievers, E. Shefler, S. Gabriel, N. Hacohen, R. Reed, M. Meyerson, T. R. Golub, E. S. Lander, D. Neuberg, J. R. Brown, G. Getz, C. J. Wu, SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. N. Engl. J. Med. 365 , 2497–2506 (2011).22150006
48 S. J. Furney, M. Pedersen, D. Gentien, A. G. Dumont, A. Rapinat, L. Desjardins, S. Turajlic, S. Piperno-Neumann, P. de la Grange, S. Roman-Roman, M. H. Stern, R. Marais, SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov. 3 , 1122–1129 (2013).23861464
49 J. W. Harbour, E. D. Roberson, H. Anbunathan, M. D. Onken, L. A. Worley, A. M. Bowcock, Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. Nat. Genet. 45 , 133–135 (2013).23313955
50 M. Martin, L. Masshofer, P. Temming, S. Rahmann, C. Metz, N. Bornfeld, J. van de Nes, L. Klein-Hitpass, A. G. Hinnebusch, B. Horsthemke, D. R. Lohmann, M. Zeschnigk, Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat. Genet. 45 , 933–936 (2013).23793026
51 A. Mupo, M. Seiler, V. Sathiaseelan, A. Pance, Y. Yang, A. A. Agrawal, F. Iorio, R. Bautista, S. Pacharne, K. Tzelepis, N. Manes, P. Wright, E. Papaemmanuil, D. G. Kent, P. C. Campbell, S. Buonamici, N. Bolli, G. S. Vassiliou, Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts. Leukemia 31 , 720–727 (2017).27604819
52 A. Castro, C. Bernis, S. Vigneron, J. C. Labbe, T. Lorca, The anaphase-promoting complex: A key factor in the regulation of cell cycle. Oncogene 24 , 314–325 (2005).15678131
53 L. Zhang, X. Zhang, H. Zhang, F. Liu, Y. Bi, Y. Zhang, C. Cheng, J. Liu, Knockdown of SF3B1 inhibits cell proliferation, invasion and migration triggering apoptosis in breast cancer via aberrant splicing. Breast Cancer 27 , 464–476 (2020).31919642
54 A. Tanaka, S. Sakaguchi, Regulatory T cells in cancer immunotherapy. Cell Res. 27 , 109–118 (2017).27995907
55 J. Liu, P. C. Lin, B. P. Zhou, Inflammation fuels tumor progress and metastasis. Curr. Pharm. Des. 21 , 3032–3040 (2015).26004407
56 L. M. Coussens, Z. Werb, Inflammation and cancer. Nature 420 , 860–867 (2002).12490959
57 C. M. Ulrich, J. Bigler, J. D. Potter, Non-steroidal anti-inflammatory drugs for cancer prevention: Promise, perils and pharmacogenetics. Nat. Rev. Cancer 6 , 130–140 (2006).16491072
58 S. Shalapour, M. Karin, Immunity, inflammation, and cancer: An eternal fight between good and evil. J. Clin. Invest. 125 , 3347–3355 (2015).26325032
59 F. Caiado, E. M. Pietras, M. G. Manz, Inflammation as a regulator of hematopoietic stem cell function in disease, aging, and clonal selection. J. Exp. Med. 218 , e20201541 (2021).34129016
60 J. J. Trowbridge, D. T. Starczynowski, Innate immune pathways and inflammation in hematopoietic aging, clonal hematopoiesis, and MDS. J. Exp. Med. 218 , e20201544 (2021).34129017
61 T. Y. Zhang, R. Dutta, B. Benard, F. Zhao, R. Yin, R. Majeti, IL-6 blockade reverses bone marrow failure induced by human acute myeloid leukemia. Sci. Transl. Med. 12 , eaax5104 (2020).32269167
62 S. Hemmati, T. Haque, K. Gritsman, Inflammatory signaling pathways in preleukemic and leukemic stem cells. Front. Oncol. 7 , 265 (2017).29181334
63 R. Radpour, C. Riether, C. Simillion, S. Hopner, R. Bruggmann, A. F. Ochsenbein, CD8+ T cells expand stem and progenitor cells in favorable but not adverse risk acute myeloid leukemia. Leukemia 33 , 2379–2392 (2019).30877275
64 D. Hormaechea-Agulla, K. A. Matatall, D. T. Le, B. Kain, X. Long, P. Kus, R. Jaksik, G. A. Challen, M. Kimmel, K. Y. King, Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNγ signaling. Cell Stem Cell 28 , 1428–1442.e426 (2021).33743191
65 C. Schinke, O. Giricz, W. J. Li, A. Shastri, S. Gordon, L. Barreryo, T. Bhagat, S. Bhattacharyya, N. Ramachandra, M. Bartenstein, A. Pellagatti, J. Boultwood, A. Wickrema, Y. T. Yu, B. Will, S. Wei, U. Steidl, A. Verma, IL8-CXCR2 pathway inhibition as a therapeutic strategy against MDS and AML stem cells. Blood 125 , 3144–3152 (2015).25810490
66 G. W. Rhyasen, L. Bolanos, J. Fang, A. Jerez, M. Wunderlich, C. Rigolino, L. Mathews, M. Ferrer, N. Southall, R. Guha, J. Keller, C. Thomas, L. J. Beverly, A. Cortelezzi, E. N. Oliva, M. Cuzzola, J. P. Maciejewski, J. C. Mulloy, D. T. Starczynowski, Targeting IRAK1 as a therapeutic approach for myelodysplastic syndrome. Cancer Cell 24 , 90–104 (2013).23845443
67 L. Barreyro, T. M. Chlon, D. T. Starczynowski, Chronic immune response dysregulation in MDS pathogenesis. Blood 132 , 1553–1560 (2018).30104218
68 G. C. Leonardi, G. Accardi, R. Monastero, F. Nicoletti, M. Libra, Ageing: From inflammation to cancer. Immun. Ageing 15 , 1 (2018).29387133
69 T. Niccoli, L. Partridge, Ageing as a risk factor for disease. Curr. Biol. 22 , R741–R752 (2012).22975005
70 B. Bottazzi, E. Riboli, A. Mantovani, Aging, inflammation and cancer. Semin. Immunol. 40 , 74–82 (2018).30409538
71 R. Bejar, Splicing factor mutations in cancer. Adv. Exp. Med. Biol. 907 , 215–228 (2016).27256388
72 S. Shen, J. W. Park, Z. X. Lu, L. Lin, M. D. Henry, Y. N. Wu, Q. Zhou, Y. Xing, rMATS: Robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc. Natl. Acad. Sci. U.S.A. 111 , 5593–5601 (2014).
