Med1 controls CD8 T cell maintenance through IL‐7R‐mediated cell survival signalling

Abstract Under steady‐state conditions, the pool size of peripheral CD8+ T cells is maintained through turnover and survival. Beyond TCR and IL‐7R signals, the underlying mechanisms are less well understood. In the present study, we found a significant reduction of CD8+ T cell proportion in spleens but not in thymi of mice with T cell‐specific deletion of Mediator Subunit 1 (Med1). A competitive transfer of wild‐type (WT) and Med1‐deficient CD8+ T cells reproduced the phenotype in the same recipients and confirmed intrinsic role of Med1. Furthermore, we observed a comparable degree of migration and proliferation but a significant increase of cell death in Med1‐deficient CD8+ T cells compared with WT counterparts. Finally, Med1‐deficient CD8+ T cells exhibited a decreased expression of interleukin‐7 receptor α (IL‐7Rα), down‐regulation of phosphorylated‐STAT5 (pSTAT5) and Bim up‐regulation. Collectively, our study reveals a novel role of Med1 in the maintenance of CD8+ T cells through IL‐7Rα/STAT5 pathway‐mediated cell survival.


| INTRODUC TI ON
CD8 + T cells, also known as cytotoxic T cells, are pivotal for the immune defence against pathogen infection and tumour growth. 1 The pool size and status of peripheral CD8 + T cells determine the power of immune response. Basal TCR signal induced by contact with self-peptide/MHC ligands is necessary for naïve T cell survival and homeostatic proliferation, as deprivation of MHC I molecules or ablating TCR expression renders naïve T cells sensitive to cell death. 2 Basal TCR signalling induces pro-survival molecules expression indirectly by augmenting responsiveness to cytokines. 3 Cytokine signals are also responsible for naïve T cell maintenance in the periphery by controlling homeostatic proliferation and survival. 4,5 Among well-known cytokines, Interleukin-7 (IL-7) is a key cytokine required for the survival and proliferation of peripheral naïve CD8 + T cells. [6][7][8] IL-7Rα forms a heterodimer with common γc chain. 9 The surface domain binds to IL-7 cytokine, while the intercellular domain bridges JAK-1 and JAK-3. Two JAK proteins phosphorylate each other and activate STAT5 downstream, as well as PI3K-Akt-mTOR and MEK-ERK pathways. 10 IL-7/IL-7Rα-mediated signals maintain T cell survival by inducing the expression of pro-survival factors Bcl2 and Mcl1, and down-regulating proapoptotic proteins Bad, Bax, Bim etc. [11][12][13][14][15] Mediator, as a large complex composed of 25-30 subunits, which is a highly conserved and integral part of RNAP II-mediated transcriptional machinery of the eukaryotes. It serves as a molecular bridge between transcription factors and the RNA polymerase II. 16 Med1, as a key component of mediator complex, is associated with the binding of a variety of cofactors to regulate gene transcription. 17 Med1 can promote cell proliferation and survival through interacting with transcription factors, such as p53, 18 GATA family proteins 19,20 and PPARα, 21 and contribute to the pathogenesis of various of cancer, including breast cancer, hepatoma and prostate cancer. [22][23][24] Med1 mutation in mice results in embryonic and early postnatal lethality. 25 It was recently reported that Med1 deletion could impair the transition of developmental stages in thymic invariant natural killer T (iNKT) cells without affecting thymic αβ T cells. 26 A reduction of CD8 + T cell population in the spleen was also mentioned. However, it is unclear whether Med1 plays an intrinsic role and what is the involved mechanism.
In the present study, we report that Med1 deletion in T cells intrinsically impaired CD8 + T cell population size in the periphery of unchallenged mice. Med1-deficient CD8 + T cells exhibited an increase in apoptosis. Mechanistically, Med1 deletion caused a reduction in IL-7Rα expression, reduction of pSTAT5 expression downstream as well as up-regulation of Bim. In summary, our study reveals Med1 is required for CD8 + T cell survival and homeostatic maintenance.

| Mice
The Med1 f/f strain was generated as previously described. 27 Med1 TKO mice (LckCre + Med1 f/f ) were generated by crossing Med1 f/f mice with LckCre transgenic strain. All mice were bred and maintained in the specific pathogen-free conditions by Xi'an Jiaotong University Division of Laboratory Animal Research. All the procedures were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

| Flow cytometry and sorting
Single cells were obtained from thymi and spleens of indicated mice.

| Quantitative PCR
Quick-RNA Microprep Kit (Zymo Research) was used to extract total RNA from purified CD8 + T cells following the manufacturer's instruction. cDNA was synthesized by the cDNA synthesis kit (TOYOBO).

| Western blotting
Purified naïve CD8+ T cells were lysed in RIPA lysis buffer (Beyotime Biotechnology) to extract the total proteins. Samples were separated on SDS-polyacrylamide gels and electro-transferred onto Polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% skimmed milk, the membranes were incubated with indicated Med1, STAT5, pSTAT5 and Bim antibodies at 4°C overnight. The membranes were then incubated with secondary antibodies at room temperature for 1h.

| Statistics
Data are presented as mean ± SEM. Statistical significance was analysed using the GraphPad Prism 7.0 statistical program. All comparisons between two experimental groups were performed with an unpaired two-tailed Student's t test. The levels of significance are indicated as follows: *P < .05, **P < .01 and ***P < .001.

| A significant reduction of CD8 + T cells in the spleen but not in the thymus of Med1-deficient mice
To study whether Med1 regulates T cell development, we gener-

| An intrinsic role of Med1 in peripheral CD8 + T cell population
To confirm the intrinsic role of Med1 in CD8 + T cell population in the spleen, we adoptively transferred a mixture of bone marrow cells from WT (CD45.2 + ) and Med1 TKO mice (CD45.1 + ) at a 1:1 ratio into lethally F I G U R E 1 The percentage and number of CD4 + and CD8 + T cells in the thymus and spleen of WT and Med1 TKO mice. Thymocytes and splenocytes from 8-week-old WT and Med1 TKO mice were stained with anti-CD4 and anti-CD8 antibodies. A, Representative FACS plots of CD4 and CD8 expression in thymocytes and splenocytes. B, The percentage and number of CD4 + thymocytes. C, The percentage and number of CD8 + thymocytes. B, The percentage and number of CD4 + splenocytes. C, The percentage and number of CD8 + splenocytes. A-D, Results shown are from one experiment representative of three independent experiments with a total of nine mice per group. *P < .05 and **P < .01 was considered statistically significant and extremely significant, respectively 11

| Reduction of CD8 + T cells in spleen is due to defect in homeostasis
To address whether the reduction of CD8 + T cell population is

| Med1 deletion enhanced apoptosis in Med1deficient CD8 + T cells
To determine the mechanism for CD8 + T cell reduction in the periphery, we evaluated proliferation and apoptosis using BrdU incorporation, Ki67 and Annexin V staining in peripheral T cells. Our data showed a low expression of BrdU and Ki67 in both CD4 + and CD8 + T cells from the spleens ( Figure 4A,D). The percentages of BrdU + and Ki67 + were similar in CD4 + T cells from WT and Med1 TKO group ( Figure 4B,E), whereas the percentages of BrdU + and Ki67 + cells were slightly higher in Med1-deficient CD8 + T cells than WT T cells ( Figure 4C,F). Importantly, we showed a significant increase of Annexin V + cells in CD8 + T cells ( Figure 4G,I), but not in CD4 + T cells from Med1 TKO in comparison with that from WT mice ( Figure 4H).
The data demonstrate that Med1 is required for CD8 + T cell survival under homeostatic conditions.

| Loss of Med1 significantly impaired IL-7Rα signalling and activated Bim expression
In the context of T cell homeostasis in periphery, IL-7Rα signalling is crucial for cell survival through activating STAT5 and suppressing apoptosis pathway. 6,7 To determine whether Med1 deletion affects IL-7Rα signalling, we assessed the expression of IL-7Rα and down-

| D ISCUSS I ON
Previous studies demonstrated that naive T cell maintenance in the periphery via physiological homeostatic mechanisms primarily relies on TCR and IL-7Rα signals. However, there is lack of detailed mechanisms underlying the maintenance of peripheral T cells. In the current study, we reported that Med1 is specifically and intrinsically required for the maintenance of CD8 + T cell pool in the spleen.
Med1 is essential for the development of several organs, including heart, eye, vascular and hematopoietic system. 28,29 Recently, it has been reported that Med1-deficiency led to a reduction in CD8 +

ACK N OWLED G EM ENTS
We thank 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
We declare no competing interests.