Pbrm1 intrinsically controls the development and effector differentiation of iNKT cells

Abstract Under static condition, the pool size of peripheral invariant natural killer T (iNKT) cells is determined by their homeostatic proliferation, survival and thymic input. However, the underlying mechanism is not fully understood. In the present study, we found that the percentage and number of iNKT cells were significantly reduced in the spleen, but not in the thymus of mice with deletion of polybromo‐1 (Pbrm1) compared to wild type (WT) mice. Pbrm1 deletion did not affect iNKT cell proliferation and survival, instead significantly impaired their development from stage 1 to stage 2. Importantly, loss of Pbrm1 led to a dysfunction of RORγt expression and iNKT17 cell differentiation, but not iNKT1 and iNKT2 proportion. Collectively, our study reveals a novel mechanism of Pbrm1 controlling the peripheral size of iNKT cells through regulating their development and differentiation.


| INTRODUC TI ON
As an "innate-like" T cells, invariant natural killer T (iNKT) cells express both T cell marker (CD3) and NK cell marker (NK1.1). 1 cells, but significantly decreased in the stage 3. Indeed, abrogation of PLZF leads to a developmental block in stage 0 of iNKT cells. 15 Along with the iNKT cell development, Egr1/2 mediates the transition from stage 1 to stage 2 through binding to the promoter of PLZF and regulating its transcription. 16 Whereas Notch1/2 signaling is involved in the progression to stage 3, as loss of Notch1/2 leads to a decreased proportion of iNKT cells at stage 3. 17 Besides, E protein, 18 Hobit 19 and T-bet 20,21 also contribute to the regulation of the stage 3 of iNKT cell development. Additionally, several other transcription factors are involved in iNKT cell survival such as c-Myc, 22 NF-κB 23 and TCF-1. 24 Based on the expression of master transcription factors, mature iNKT cells can be categorized into three functional subsets, namely iNKT1, iNKT2 and iNKT17. 25 PLZF, with varied expression level among iNKT subsets, is also essential for their functional differentiation. 15 Compared to iNKT2 cells, which express the largest amount of PLZF, IFNγ-secreting iNKT1 cells express much lower amount of PLZF. Whereas, the expression level of PLZF in iNKT17 cells is between iNKT1 and iNKT2 cells. 26 As the traditional T helper cell-specific transcription factors, T-box 21 (T-bet), GATA binding protein-3 (GATA3) and retinoic acid receptor-related orphan nuclear receptor gamma (RORγt), also regulate the iNKT lineage decisions. [27][28][29][30] Loss of T-bet exhibits a block in the differentiation of iNKT1 along with decreased interferonγ (IFNγ) production. 21 Furthermore, GATA3 expression is essential for iNKT2 cells differentiation and IL-4 production, 28 while RORγt, 29,30 combined with other factors like E protein, 18 is involved in iNKT17 cell differentiation. Nevertheless, there are still largely unknown regulators for iNKT cell development in the thymus and their effector differentiation in the periphery.
Pbrm1, as a subunit of the Pbrm1-Brg1/Brm-associated factors (PBAF) complex, interacts with ARID2, BRD7, BAF45A, as well as several other subunits to form a SWI/SNF chromatin remodeling complex. [31][32][33] As a tumor suppressor, Pbrm1 is mutated frequently in diverse cancer types, and its reduced expression is positively correlated with the tumor progression. [34][35][36][37] Pbrm1 has also been reported to be involved in regulating T cell development and differentiation. Although Pbrm1 deficiency did not strongly affect the thymocyte development and the proportion of peripheral T cells, 38 loss of Brg1 exhibited a delay of CD8 expression in DP cells, suggesting a favorable role for CD8 transcription. 39,40 In addition, Brg1 is required for T helper differentiation including Th1 41 and   Th2 42 since loss of Brg1 caused a reduction in both IFNγ and IL-4 production under the polarized condition. Other studies reported that Pbrm1 is a repressor of IL-10 transcription in Th2 cells by either binding directly to the regulatory region in the Il-10 locus, or regulating histone acetylation and CBP recruitment to the IL-10 locus. 38 A recent study also showed that Pbrm1 deletion caused a downregulation of foxp3 expression and an impairment in Treg suppressive function. 32 Yet, the function of Pbrm1 in other lineage of immune cells is largely unknown.
In the present study, by utilizing mice with T-cell-specific ablation of Pbrm1 and in vivo adoptive transfer model, we demonstrated that Pbrm1 deficiency did not affect CD4 + and CD8 + T cell proportions in the thymus and spleen. However, Pbrm1-deficient iNKT cells were significantly reduced in the spleen, as Pbrm1 specifically blocked the transition of iNKT cell development from stage 1 to stage 2 in the thymus. Furthermore, Pbrm1-deficient mice exhibited an impairment of RORγt expression and iNKT17 lineage differentiation. Therefore, our study reveals a novel role of Pbrm1 in regulating the development and differentiation of iNKT cells.

| Mice
The Pbrm1 f/f strain was purchased from The Jackson Laboratory.

| Antibodies and reagents
The Abs used are as follows: APC/Cy7 anti-mouse CD4 (clone    For apoptosis analysis, freshly-isolated cells were stained with the antibodies for indicated surface markers. After wash with FACS buffer, apoptosis was evaluated with Annexin V as previously described. 43 To analyze intracellular transcriptional factors, after a 30 min surface staining, cells were fixed and permeabilized according to the manual of the Foxp3 kit, followed by anti-RORγt and anti-PLZF antibody staining and FACS analysis.

| Statistical analysis
Statistical analysis was applied to biologically independent mice or technical replicates for each experiment. Each experiment was independently repeated for three times. The two-tailed Student's t test was used for all statistical calculations using GraphPad Prism 7 software. The level of significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

| A reduction of peripheral iNKT cells in Pbrm1 deficient mice
To  (Figure 2A-C). Together, we demonstrated that Pbrm1 is required for the population size of peripheral iNKT cells.

| Pbrm1 deletion has no impact in the proliferation and survival of iNKT cells
We then investigated whether the reduction of iNKT cells in the pe-

| Loss of Pbrm1 caused a differentiation block at stage 1 iNKT cells
We next sought to determine whether the decrease of iNKT cells was  Figure 4A,B). Consistently, as shown in Figure 4C,D, the dysfunction of iNKT cells in Pbrm1-deficient mice was further confirmed in bone marrow chimera mice. Together, the reduced number of iNKT cells in Pbrm1 KO mice is most likely due to a developmental block from stage 1 to stage 2/3.

| DISCUSS ION
In the current study, we reveal an intrinsic role of Pbrm1, as a subunit   The distinct effector subsets of iNKT cells endow their potent protective immunity and immune tolerance. 47 It was reported that SRG3, as a components of SWI/SNF complex could assist Th17 cell differentiation through collaboration with RORγt. 48 Consistently, our data showed that iNKT17 subset, but not iNKT1 and iNKT2 cells,

ACK N OWLED G EM ENT
We thank Prof. Enqi Liu from Laboratory Animal Center for mouse colony maintenance, and Prof. Chen Huang and Dr. Xiaofei Wang from Department of Cell Biology and Genetics for flow cytometric analysis and cell-sorting.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data and models during the study are available in the submitted article.