ARTICLE DOI: 10.1038/s41467-017-00348-3 OPEN The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity Woelsung Yi1, Sanjay Gupta1, Edd Ricker2, Michela Manni1, Rolf Jessberger3, Yurii Chinenov4,5, Henrik Molina6 & Alessandra B. Pernis1,2,7 Post-transcriptional modifications can control protein abundance, but the extent to which these alterations contribute to the expression of T helper (TH) lineage-defining factors is unknown. Tight regulation of Bcl6 expression, an essential transcription factor for T follicular helper (TFH) cells, is critical as aberrant TFH cell expansion is associated with autoimmune diseases, such as systemic lupus erythematosus (SLE). Here we show that lack of the SLE risk variant Def6 results in deregulation of Bcl6 protein synthesis in T cells as a result of enhanced activation of the mTORC1–4E-BP–eIF4E axis, secondary to aberrant assembly of a raptor–p62–TRAF6 complex. Proteomic analysis reveals that this pathway selectively controls the abundance of a subset of proteins. Rapamycin or raptor deletion ameliorates the aberrant TFH cell expansion in mice lacking Def6. Thus deregulation of mTORC1-dependent pathways controlling protein synthesis can result in T-cell dysfunction, indicating a mechanism by which mTORC1 can promote autoimmunity. 1 Autoimmunity and Inflammation Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York 10021 USA. 2 Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, 1300 York Avenue, Box 65, New York, New York 10021 USA. 3 Institute of Physiological Chemistry, Technische Universität Dresden, Fiedlerstrasse 42, MTZ, 01307 Dresden, Germany. 4 Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, 535 East 70th Street, New York, New York 10021 USA. 5 David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, 535 East 70th Street, New York, New York 10021 USA. 6 Proteomics Resource Center, The Rockefeller University, 1230 York Avenue, Box 105, New York, New York 10065 USA. 7 Department of Medicine, Weill Cornell Medical College, Cornell University, 525 East 68th Street, Box 130, New York, New York 10065 USA. Sanjay Gupta and Edd Ricker contributed equally to this work. Correspondence and requests for materials should be addressed to A.B.P. (email: [email protected]) NATURE COMMUNICATIONS | 8: 254 | DOI: 10.1038/s41467-017-00348-3 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00348-3 recise regulation of T follicular helper (TFH) cell numbers is differentiation, indicating that tight control of Bcl6 expression is Pcritical for optimal humoral responses, and aberrant essential to ensure proper regulation of TFH cell numbers. Bcl6 expansion of TFH cells is associated with autoimmune dis- expression in TFH cells has, until now, been shown to be primarily eases, including systemic lupus erythematosus (SLE)1, 2. The regulated by transcriptional mechanisms6. The expression of transcriptional repressor Bcl6 is a lineage-defining factor for TFH Bcl6, however, can be controlled by complex regulatory networks 3–5 fi cells . Bcl6 is necessary to specify the TFH cell program and that ne-tune Bcl6 expression by targeting both Bcl6 mRNA and 7 overexpression of Bcl6 is sufficient to drive TFH cell protein . In B cells, Bcl6 levels are regulated by a number of post- abcd6–11 weeks old 6–11 weeks old >24 weeks old >24 weeks old WT DKO WT DKO 15 P < 0.0001 P < 0.0001 ) hi 30 ) + hi 1.9 8.5 4.0 23.9 + PD1 10 PD1 20 hi hi 5 10 PD1 PD1 CXCR5 cells (% CD4 CXCR5 0 cells (% CD4 0 WT DKO WT DKO CXCR5 CXCR5 3 P < 0.0001 15 P < 0.0001 hi hi 3.8 ) 1.6 5.7 ) 15.0 6 6 PD1 PD1 2 10 hi hi 1 5 cells (×10 Bcl6 cells (×10 Bcl6 CXCR5 CXCR5 0 0 WT DKO WT DKO CXCR5 CXCR5 eftr/tr –/– 40 P < 0.0001 WT Def6 Swap-70 DKO ) hi + P = 0.0046 30 3.65 18.6 3.54 23.9 P = 0.0006 PD1 hi 20 10 CXCR5 PD1 cells (% CD4 0 WT Def6tr/tr Swap-70–/– DKO CXCR5 20 P = 0.0002 4.31 15.9 4.21 15 hi ) P = 0.0172 6 15 PD1 hi P = 0.0474 10 Bcl6 5 cells (×10 CXCR5 0 CXCR5 WT Def6tr/tr Swap-70–/– DKO g 6–11 weeks old WT WT (NP-CGG) DKO PNA PD1 PNA CD3 hi>24 weeks old >24 weeks old + + hi hi + + hi hi CD4 CD4 CXCR5 PD1 ) CD4 CD4 CXCR5 PD1 ) 6 6 20 P < 0.0001 10 P < 0.0001 0.3 P < 0.0345 0.3 15 8 0.2 0.2 6 cells (×10 cells (×10 + + 10 γ 4 0.1 0.1 5 IL-17 IFN- 2 + + 0 0 0.0 0.0 IL-21 IL-21 WT DKO WT DKO WT DKO WT DKO 2 NATURE COMMUNICATIONS | 8: 254 | DOI: 10.1038/s41467-017-00348-3 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00348-3 ARTICLE transcriptional mechanisms, which control Bcl6 protein stability in female mice as in human SLE21. Autoimmunity in DKO mice and its activity7. results from dual abnormalities in T and B cells, whereby the lack Among post-transcriptional mechanisms, translational control of Def6 alone is responsible for the T-cell abnormalities, and the has a major function in regulating protein abundance and can absence of both Def6 and SWAP70 contributes to the deregulated influence protein levels to an extent similar to transcription8.A B-cell responses21. critical controller of protein synthesis is mammalian target of In this study, we demonstrate that the robust humoral auto- − − rapamycin (mTOR), a serine/threonine kinase that exists in two immune responses observed in Def6tr/trSwap70 / DKO mice are distinct complexes, mTORC1 and mTORC2, distinguished by the accompanied by cell-intrinsic expansion of the TFH cell com- presence of unique components such as raptor and rictor, partment. Importantly, we show that DKO T cells have aberrant respectively9, 10. mTORC1 activation occurs in response to control of Bcl6 protein synthesis, which occurs in an mTORC1 diverse environmental cues, including growth factors, energy and eukaryotic initiation factor 4E (eIF4E)-dependent manner. status, and amino-acid availability. Growth factors activate Enhanced mTORC1 activation in DKO T cells is a result of mTORC1 mainly through the phosphoinositide-3 kinase (PI3K)- dysregulated interaction of raptor with both p62 and TRAF6, AKT pathway, whereas the energy status of a cell regulates critical regulators of an amino-acid-sensing pathway of mTORC1 mTORC1 activation via AMP-activated protein kinase activation12, 13. We also demonstrate that Def6 controls the – (AMPK)9 11. mTORC1 activation by PI3K-AKT and AMPK assembly of the raptor–p62–TRAF6 complex and that this – occurs via the TSC complex and the small GTPAse Rheb9 11.By pathway selectively regulates the abundance of a specific subset of contrast, amino acids regulate a different set of GTPases, the Rag proteins. Consistent with these findings, rapamycin administra- proteins, which recruit mTORC1 to the lysosomes enabling tion or T-cell deletion of raptor significantly decreased the subsequent activation by Rheb. Although activation of the Rags accumulation of TFH cells in DKO mice. Thus abnormalities in normally depends on their interaction with the Ragulator com- the mechanisms by which mTORC1 regulates protein abundance plex, an alternative docking system that depends on the central can result in T-cell dysfunction and contribute to autoimmunity. – signaling hub p62 can also control activation11 13. p62 interacts with and activates the Rags, helps recruit mTORC1 to the lyso- somes by binding Raptor and also mediates the assembly of a Results trimolecular complex with TRAF6, which can then activate Spontaneous expansion of TFH cells in lupus-prone DKO mice. 12, 13 mTOR kinase activity via K63-linked polyubiquitination . Precise control of TFH cell differentiation is essential to prevent 1, 2 mTOR is a major coordinator of TH cell fate decisions and SLE . Mice lacking Def6 and Swap70 (here termed DKO mice) 9, 10 regulates the differentiation of several TH subsets . mTOR spontaneously develop a systemic autoimmune disorder char- plays a complex role in TFH differentiation. Whereas the inter- acterized by increased numbers of effector T cells, germinal center leukin (IL)-2–mTORC1 axis shifted differentiation away from (GC) B cells, plasma cells, and autoantibody production21. This TFH cells toward the TH1 lineage in an acute viral infection robust humoral autoimmune response led us to speculate that model14, mTORC1 activation is required for the spontaneous many of the effector T cells that accumulate in these mice might ’ formation of TFH cells in Peyer s patches and for the induction of be TFH cells. A detailed analysis indeed revealed that young DKO 15, 16 TFH cells upon immunization with a foreign antigen . mice spontaneously display increased frequencies and numbers of hi hi + mTORC2 activity is also important for TFH differentiation, par- TFH cells based on staining for either CXCR5 PD1 CD4 ’ 16 hi hi + ticularly in Peyer s patches . The varying requirements of TFH T cells or CXCR5 Bcl6 CD4 T cells (Figs. 1a, b). Similar cells on mTOR activity are probably due to differences in the results were also obtained when TFR cells, a subset of 16 hi hi precise environmental cues to which TFH cells are exposed . CXCR5 PD1 cells that expresses Foxp3 and specialize in the 22, 23 mTOR has been shown to regulate TH cell differentiation by inhibition of the GC reaction , was excluded from the analysis controlling the transcription of master regulators and metabolic by gating out Foxp3+ cells (Supplementary Figs. 1a, b, e, f). DKO reprogramming. Although regulation of protein synthesis is also a TFH cells further expanded with age (Figs.