The kinase PDK1 is a crucial regulator for immune cell development by connecting PI3K to downstream AKT signaling. However, the roles of PDK1 in CD4
T cell differentiation, especially in T follicular ...helper (Tfh) cell, remain obscure. Here we reported PDK1 intrinsically promotes the Tfh cell differentiation and germinal center responses upon acute infection by using conditional knockout mice. PDK1 deficiency in T cells caused severe defects in both early differentiation and late maintenance of Tfh cells. The expression of key Tfh regulators was remarkably downregulated in PDK1-deficient Tfh cells, including
,
,
, and
. Mechanistically, ablation of PDK1 led to impaired phosphorylation of AKT and defective activation of mTORC1, resulting in substantially reduced expression of Hif1α and p-STAT3. Meanwhile, decreased p-AKT also suppresses mTORC2-associated GSK3β activity in PDK1-deficient Tfh cells. These integrated effects contributed to the dramatical reduced expression of TCF1 and ultimately impaired the Tfh cell differentiation.
N6-methyladenosine (m6A) methyltransferase Mettl3 is involved in conventional T cell immunity; however, its role in innate immune cells remains largely unknown. Here, we show that Mettl3 ...intrinsically regulates invariant natural killer T (iNKT) cell development and function in an m6A-dependent manner. Conditional ablation of Mettl3 in CD4+CD8+ double-positive (DP) thymocytes impairs iNKT cell proliferation, differentiation, and cytokine secretion, which synergistically causes defects in B16F10 melanoma resistance. Transcriptomic and epi-transcriptomic analyses reveal that Mettl3 deficiency disturbs the expression of iNKT cell-related genes with altered m6A modification. Strikingly, Mettl3 modulates the stability of the Creb1 transcript, which in turn controls the protein and phosphorylation levels of Creb1. Furthermore, conditional targeting of Creb1 in DP thymocytes results in similar phenotypes of iNKT cells lacking Mettl3. Importantly, ectopic expression of Creb1 largely rectifies such developmental defects in Mettl3-deficient iNKT cells. These findings reveal that the Mettl3-m6A-Creb1 axis plays critical roles in regulating iNKT cells at the post-transcriptional layer.
Display omitted
•Mettl3-dependent m6A methylation plays a key role in iNKT cell development•A comprehensive m6A landscape of low-input iNKT cells is established by m6A-LACE-seq•Mettl3 deficiency disrupts iNKT cell-related gene network in an m6A-dependent manner•The Mettl3-m6A-Creb1 axis is critical for sustaining iNKT cell differentiation and function
You et al. demonstrate that Mettl3 is essential for regulating iNKT cell proliferation, differentiation, and melanoma resistance. The study highlights the critical role of Mettl3-dependent m6A modification in controlling iNKT cell-related gene expression, particularly in maintaining the stability of Creb1 and c-Myc transcripts.
Invariant natural killer T (iNKT) cells are highly conserved innate-like T lymphocytes that originate from CD4
CD8
double-positive (DP) thymocytes. Here, we report that serine/arginine splicing ...factor 1 (SRSF1) intrinsically regulates iNKT cell development by directly targeting Myb and balancing the abundance of short and long isoforms. Conditional ablation of SRSF1 in DP cells led to a substantially diminished iNKT cell pool due to defects in proliferation, survival, and TCRα rearrangement. The transition from stage 0 to stage 1 of iNKT cells was substantially blocked, and the iNKT2 subset was notably diminished in SRSF1-deficient mice. SRSF1 deficiency resulted in aberrant expression of a series of regulators that are tightly correlated with iNKT cell development and iNKT2 differentiation, including Myb, PLZF, Gata3, ICOS, and CD5. In particular, we found that SRSF1 directly binds and regulates pre-mRNA alternative splicing of Myb and that the expression of the short isoform of Myb is substantially reduced in SRSF1-deficient DP and iNKT cells. Strikingly, ectopic expression of the Myb short isoform partially rectified the defects caused by ablation of SRSF1. Furthermore, we confirmed that the SRSF1-deficient mice exhibited resistance to acute liver injury upon α-GalCer and Con A induction. Our findings thus uncovered a previously unknown role of SRSF1 as an essential post-transcriptional regulator in iNKT cell development and functional differentiation, providing new clinical insights into iNKT-correlated disease.
Abstract
T follicular helper (T
FH
) cells are specialized effector CD4
+
T cells critical to humoral immunity. Whether post-transcriptional regulation has a function in T
FH
cells is unknown. Here, ...we show conditional deletion of METTL3 (a methyltransferase catalyzing mRNA
N
6
-methyladenosine (m
6
A) modification) in CD4
+
T cells impairs T
FH
differentiation and germinal center responses in a cell-intrinsic manner in mice. METTL3 is necessary for expression of important T
FH
signature genes, including
Tcf7
,
Bcl6
,
Icos
and
Cxcr5
and these effects depend on intact methyltransferase activity. m
6
A-miCLIP-seq shows the 3′ UTR of
Tcf7
mRNA is subjected to METTL3-dependent m
6
A modification. Loss of METTL3 or mutation of the
Tcf7
3′ UTR m
6
A site results in accelerated decay of
Tcf7
transcripts. Importantly, ectopic expression of TCF-1 (encoded by
Tcf7
) rectifies T
FH
defects owing to METTL3 deficiency. Our findings indicate that METTL3 stabilizes
Tcf7
transcripts via m
6
A modification to ensure activation of a T
FH
transcriptional program, indicating a pivotal function of post-transcriptional regulation in promoting T
FH
cell differentiation.
The underlying mechanisms of thymocyte development and lineage determination remain incompletely understood, and the emerging evidences demonstrated that RNA binding proteins (RBPs) are deeply ...involved in governing T cell fate in thymus. Serine/arginine-rich splicing factor 1 (SRSF1), as a classical splicing factor, is a pivotal RBP for gene expression in various biological processes. Our recent study demonstrated that SRSF1 plays essential roles in the development of late thymocytes by modulating the T cell regulatory gene networks post-transcriptionally, which are critical in response to type I interferon signaling for supporting thymocyte maturation. Here, we report SRSF1 also contributes to the determination of the CD8
T cell fate. By specific ablation of SRSF1 in CD4
CD8
double positive (DP) thymocytes, we found that SRSF1 deficiency impaired the maturation of late thymocytes and diminished the output of both CD4
and CD8
single positive T cells. Interestingly, the ratio of mature CD4
to CD8
cells was notably altered and more severe defects were exhibited in CD8
lineage than those in CD4
lineage, reflecting the specific function of SRSF1 in CD8
T cell fate decision. Mechanistically, SRSF1-deficient cells downregulate their expression of
, which is a crucial transcriptional regulator in sustaining CD8
single positive (SP) thymocyte development and lineage choice. Moreover, forced expression of Runx3 partially rectified the defects in SRSF1-deficient CD8
thymocyte maturation. Thus, our data uncovered the previous unknown role of SRSF1 in establishment of CD8
cell identity.
ABSTRACTHematopoietic stem cells (HSCs) have the capacity for self‐renewal to maintain the HSCs' pool and the ability for multilineage differentiation, which are responsible for sustained production ...of multiple blood lineages. The regulation of HSC development is controlled precisely by complex signal networks and hematopoietic microenvironment, which has been termed the HSCs' niche. The Wnt signaling pathway is one of a variety of signaling pathways that have been involved in HSC self‐renewal and maintenance. Previous studies are indeterminant on the regulation of adult HSCs upon canonical Wnt signaling pathways because of the different experimental systems and models used. In this study, we generated the conditional knockout Wnt coreceptor low‐density lipoprotein receptor‐related protein 5 (Lrp5) and low‐density lipoprotein receptor‐related protein 6 (Lrp6) mice in adult hematopoiesis via Vav‐Cre Loxp system. Inactivation of Lrp5 and ‐6 in a hematopoietic system diminished the pool of HSCs, but there were no obvious defects in mature immune cells. Lrp5 and ‐6 double deficiency HSCs showed intrinsic defects in self‐renewal and differentiation due to reduced proliferation and increased quiescence of the cell cycle. Analysis of HSC gene expression suggested that the quiescence regulators were significantly up‐regulated, such as Egr1, Cdkn1a, Nr4a1, Gata2, Junb and Btg2, and the positive cell cycle regulators were correspondingly down‐regulated, such as Ccna2 and Ranbp1. Taken together, we investigated the roles of Lrp5 and ‐6 in HSCs by functional and bioinformatic assays, and we demonstrated that Lrp5 and ‐6 are required for the self‐renewal and differentiation of adult HSCs. The canonical Wnt pathway may contribute to maintaining the HSC pool and regulate the differentiation of adult HSCs by controlling cell cycle gene regulatory module.—Liu, J., Cui, Z., Wang, F., Yao, Y., Yu, G., Liu, J., Cao, D., Niu, S., You, M., Sun, Z., Lian, D., Zhao, T., Kang, Y., Zhao, Y., Xue, H.‐H., Yu, S. Lrp5 and Lrp6 are required for maintaining self‐renewal and differentiation of hematopoietic stem cells. FASEB J. 33, 5615–5625 (2019). www.fasebj.org
Long noncoding RNAs (lncRNAs) are emerging as critical mediators of various biological processes in the immune system. The current data showed that the lncRNA Malat1 is highly expressed in T cell ...subsets, but the function of Malat1 in T cell remains unclear. In this study, we detected the T cell development and both CD8
+
and CD4
+
T cell response to LCMV infection using Malat1
−/-
mice model. To our surprise, there were no significant defects in thymocytes at different developmental stages and the peripheral T cell pool with ablation of Malat1. During LCMV infection, Malat1
−/-
mice exhibited normal effector and memory CD8
+
T cells as well as T
FH
cells differentiation. Our results indicated that Malat1 is not essential for T cell development and T cell-mediated antiviral response though it expresses at very high level in different T cell populations.
The underlying mechanisms of thymocyte maturation remain largely unknown. Here, we report that serine/arginine-rich splicing factor 1 (SRSF1) intrinsically regulates the late stage of thymocyte ...development. Conditional deletion of SRSF1 resulted in severe defects in maintenance of late thymocyte survival and a blockade of the transition of TCRβ
CD24
CD69
immature to TCRβ
CD24
CD69
mature thymocytes, corresponding to a notable reduction of recent thymic emigrants and diminished periphery T cell pool. Mechanistically, SRSF1 regulates the gene networks involved in thymocyte differentiation, proliferation, apoptosis, and type I interferon signaling pathway to safeguard T cell intrathymic maturation. In particular, SRSF1 directly binds and regulates
and
expression via alternative splicing in response to type I interferon signaling. Moreover, forced expression of interferon regulatory factor 7 rectifies the defects in SRSF1-deficient thymocyte maturation via restoring expression of type I interferon-related genes. Thus, our work provides new insight on SRSF1-mediated posttranscriptional regulatory mechanism of thymocyte development.
PDF
