SerpinB1 controls encephalitogenic T helper cells in neuroinflammation Lifei Houa,b,1,2, Deepak A. Raoc,d, Koichi Yukie,f, Jessica Cooleya, Lauren A. Hendersonb,g, A. Helena Jonssonc,d, Dion Kaisermanh, Mark P. Gormanb,i, Peter A. Nigrovicc,d,g, Phillip I. Birdh, Burkhard Becherj, and Eileen Remold-O’Donnella,b,k,2 aThe Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115; bDepartment of Pediatrics, Harvard Medical School, Boston, MA 02115; cDivision of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Boston, MA 02115; dDepartment of Medicine, Harvard Medical School, Boston, MA 02115; eDepartment of Anesthesiology, Critical Care and Pain Medicine, Boston Children’s Hospital, Boston, MA 02115; fDepartment of Anesthesiology, Harvard Medical School, Boston, MA 02115; gDivision of Immunology, Boston Children’s Hospital, Boston, MA 02115; hDepartment of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, 3800, Australia; iDepartment of Neurology, Boston Children’s Hospital, Boston, MA 02115; jInflammation Unit, Institute of Experimental Immunology, University of Zurich, CH-8057 Zurich, Switzerland; and kDepartment of Hematology/Oncology, Harvard Medical School, Boston, MA 02115 Edited by Jean Laurent-Casanova, Rockefeller University, New York, NY, and approved August 27, 2019 (received for review April 5, 2019) SerpinB1, a protease inhibitor and neutrophil survival factor, was flammatory tissue injury and neutrophil death, and in naïve mice, recently linked with IL-17–expressing T cells. Here, we show that preserves the bone marrow reserve of mature neutrophils by serpinB1 (Sb1) is dramatically inducedinasubsetofeffector restricting spontaneous cell death mediated by the granule serine CD4 cells in experimental autoimmune encephalomyelitis (EAE). proteases cathepsin G and proteinase-3 (32–35). Recently, we and −/− Despite normal T cell priming, Sb1 mice are resistant to EAE others demonstrated that Sb1 selectively restricts expansion of IL- with a paucity of T helper (TH) cells that produce two or more of 17–expressing γδ T cells (36) and NK T cells (37), findings that led γ the cytokines, IFN , GM-CSF, and IL-17. These multiple cytokine- us to study adaptive Th cell development where we identified Sb1 producing CD4 cells proliferate extremely rapidly; highly express as a signature gene of TH17 cells (38). the cytolytic granule proteins perforin-A, granzyme C (GzmC), Here, we report that Sb1 expression is required for optimal and GzmA and surface receptors IL-23R, IL-7Rα,andIL-1R1;and − − development of paralysis in MOG-immunized mice. We identi- INFLAMMATION can be identified by the surface marker CXCR6. In Sb1 / mice, + fied a highly selective subset of IFNγ- and GM-CSF–expressing IMMUNOLOGY AND CXCR6 TH cells are generated but fail to expand due to en- + hanced granule protease-mediated mitochondrial damage lead- IL17 Sb1-dependent CD4 cells in the periphery of immunized ing to suicidal cell death. Finally, anti-CXCR6 antibody treatment, mice at onset of disease. We describe here isolation of these Sb1- like Sb1 deletion, dramatically reverts EAE, strongly indicating dependent primed T cells and their molecular and functional + that the CXCR6 T cells are the drivers of encephalitis. Significance serpins | multiple sclerosis | inflammatory arthritis | pathogenic T helper cells | autoimmune The study addresses a basic immunology topic with considerable clinical relevance, namely the nature of the pathogenic T helper ultiple sclerosis (MS) and murine experimental autoim- cells that drive autoimmune disorders like multiple sclerosis (MS) Mmune encephalomyelitis (EAE) are chronic demyelinating and inflammatory arthritis. Using mouse MS, we identify pre- disorders of the central nervous system driven by self-reactive TH cursors of the encephalitogenic/pathogenic T cells based on their cells (1). The disease-inducing autoimmune T cells, which are dependence on the protease inhibitor SerpinB1. Newly identified present at low numbers in the periphery and as expanded pop- signature genes reveal the unusual nature of these T cells that ulations in the central nervous system (CNS), were initially thought combine (i) inflammatory cytokine secretion, (ii) a cytolytic sys- tem, and (iii) extreme rapid proliferation. We demonstrate that to be TH1 cells because disease is abrogated by deletion of the interleukin (IL)-12 subunit p40 (2, 3). With the discoveries that their survival/expansion depends on SerpinB1 and involves reg- p40 is also a subunit of IL-23 and IL-23 plays a pivotal role in ulation of a proliferation-associated protease-mediated cell sui- cide mechanism. Importantly, we discovered that cell surface mediating disease (4–7), MS was reinterpreted as TH17-driven (8, CXCR6 is an exquisite marker of pathogenic T helper cells and 9). More recent studies established that TH17 cells themselves are not pathogenic but are converted in vivo under the priming of demonstrated that anti-CXCR6 treatment has potential to pre- myeloid cell-derived IL-1β and IL-23 into pathogenic (encephali- vent or mitigate MS. togenic) TH cells, the true drivers of disease (4, 10–14). These cells γ Author contributions: L.H., D.A.R., K.Y., M.P.G., B.B., and E.R.-O. designed research; L.H., produce interferon (IFN) and granulocyte-macrophage colony- J.C., and D.K. performed research; L.H., D.A.R., K.Y., L.A.H., A.H.J., D.K., P.A.N., and P.I.B. stimulating factor (GM-CSF) (10, 15–18), the latter required for contributed new reagents/analytic tools; L.H. and E.R.-O. analyzed data; and L.H., P.A.N., encephalitogenicity (16–18). Despite the importance of the en- P.I.B., B.B., and E.R.-O. wrote the paper. cephalitogenic TH cells, little else is known about their nature or Conflict of interest statement: L.H. and E.R.-O. filed a provisional patent related to the the factors and pathway that drive their development. findings of this study and have equity ownership and are on the Scientific Advisory Board of the recently launched Edelweiss Immune, Inc. The other authors declare no conflict In cytolytic CD8 cells and NK cells, powerful granule serine of interest. proteases that are regulated by endogenous inhibitors called serpins This article is a PNAS Direct Submission. play pivotal roles in immune surveillance against tumors and viral infection while simultaneously maintaining immune homeo- Published under the PNAS license. 1 stasis (19–26). Whether analogous granzyme-serpin regulation Present addresses: Department of Anesthesiology, Critical Care and Pain Medicine, Boston Children’s Hospital and Department of Anesthesiology, Harvard Medical School, Boston, exists in CD4 cells is not known. SerpinB1 (Sb1), previously called MA 02115. MNEI (monocyte/neutrophil elastase inhibitor), is an ancestral 2To whom correspondence may be addressed. Email: [email protected] or member of the superfamily of serpins (SERine Protease Inhibi- [email protected]. tors). It is a highly efficient inhibitor of elastinolytic and chymo- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tryptic proteases that has been best studied in neutrophils (27–31). 1073/pnas.1905762116/-/DCSupplemental. For example, in bacterial lung infection, Sb1 protects against in- First Published September 23, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1905762116 PNAS | October 8, 2019 | vol. 116 | no. 41 | 20635–20643 Downloaded by guest on September 23, 2021 signature and developmental pathway in EAE, and we demon- strate that these are the T helper cells responsible for disease. Results Sb1 Is Highly Expressed in TH Cells in EAE. Previously, we identified Sb1 as preferentially expressed in TH17 cells by studying 129S6 strain mouse cells in vitro. In preparation for working with the EAE model, we polarized naïve CD4 cells of C57BL/6 mice to TH17 cells driven by IL-6 and TGFβ (Fig. 1A and SI Ap- pendix,Fig.S1A) and we confirmed select expression of Sb1, consistent with previous findings (38) (Fig. 1A). We then showed that Sb1 is also dramatically up-regulated in vivo along with Rorc and Il17a in effector (CD44hi)CD4cellsduringEAE development (Fig. 1B). To date, the factors controlling ex- pression of Sb1 in TH cells are unknown. Online gene arrays for mice deleted for the Th17 inducer serine/threonine protein kinase (Sgk)-1 (39) revealed that Sb1 is among the top down-regulated genes in IL23-stimulated Sgk1-deficient TH17 cells, suggesting a correlation between Il23r and Sb1 in TH17 cells. To investigate this putative link, we generated mice with Il23r deleted in CD4 cells Δ (Il23r CD4). We then compared the transcriptome of wt and Δ Il23r CD4 effector (CD44hiCD62Llo) CD4 cells from the lymph node (LN) of MOG-immunized mixed chimeric mice at onset of Δ EAE. Surprisingly, wt and Il23r CD4 effector CD4 cells were not very different at the transcriptome level (Fig. 1C), and only few Fig. 2. CD4 cell autonomous deficiency of Sb1 ameliorates EAE. (A–C) Wt and − − Sb1 / mice were immunized with MOG/CFA to induce EAE. (A)Meanclinical − − score (Left) and body weight (Right)ofwt (n = 13) and Sb1 / (n = 14) mice. Experiment was repeated more than 5 times with the same pattern. (B)Spinal cord infiltrates on day 10 analyzed by FACS. n = 4–5 mice in each genotype, shown are representative of five experiments. (C) Relative gene expression of spinal infiltrates analyzed by qRT-PCR. Data represent mean of four biological replicates, each with pooled cells from 2 to 3 mice per genotype. (D)Adoptive − − transfer EAE. Wt or Sb1 / T cells from MOG-immunized mice were expanded ex vivo and transferred to naive wt or Sb1−/− recipients. Mean clinical scores for 6miceeachgenotype.(E) Naive CD4 cell transfer EAE. Wt or Sb1−/− naïve − CD4 cells were transferred to Rag1/ mice, which were then MOG-immunized to induce EAE. Mean clinical scores for 6 mice each genotype. (F)DTHresponse − − of wt and Sb1 / mice to challenge in the footpad with MOG or vehicle on day − − 6 after MOG immunization.