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IL ‐1 signaling is critical for expansion but not generation of autoreactive GM ‐ CSF + Th17 cells

2016; Springer Nature; Volume: 36; Issue: 1 Linguagem: Inglês

10.15252/embj.201694615

1460-2075

Ilgiz A. Mufazalov, Carsten Schelmbauer, Tommy Regen, Janina Kuschmann, Florian Wanke, Laureen A. Gabriel, Judith Hauptmann, Werner Müller, Emmanuel Pinteaux, Florian C. Kurschus, Ari Waisman,

Immunotherapy and Immune Responses

Article8 November 2016free access Transparent process IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells Ilgiz A Mufazalov Corresponding Author Ilgiz A Mufazalov [email protected] orcid.org/0000-0001-9332-0131 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Carsten Schelmbauer Carsten Schelmbauer Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Tommy Regen Tommy Regen Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Janina Kuschmann Janina Kuschmann Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Florian Wanke Florian Wanke Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Laureen A Gabriel Laureen A Gabriel Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Judith Hauptmann Judith Hauptmann Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Werner Müller Werner Müller orcid.org/0000-0002-1297-9725 Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Search for more papers by this author Emmanuel Pinteaux Emmanuel Pinteaux Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Search for more papers by this author Florian C Kurschus Florian C Kurschus Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Ari Waisman Corresponding Author Ari Waisman [email protected] orcid.org/0000-0003-4304-8234 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Ilgiz A Mufazalov Corresponding Author Ilgiz A Mufazalov [email protected] orcid.org/0000-0001-9332-0131 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Carsten Schelmbauer Carsten Schelmbauer Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Tommy Regen Tommy Regen Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Janina Kuschmann Janina Kuschmann Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Florian Wanke Florian Wanke Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Laureen A Gabriel Laureen A Gabriel Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Judith Hauptmann Judith Hauptmann Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Werner Müller Werner Müller orcid.org/0000-0002-1297-9725 Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Search for more papers by this author Emmanuel Pinteaux Emmanuel Pinteaux Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Search for more papers by this author Florian C Kurschus Florian C Kurschus Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Ari Waisman Corresponding Author Ari Waisman [email protected] orcid.org/0000-0003-4304-8234 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Author Information Ilgiz A Mufazalov *,1, Carsten Schelmbauer1, Tommy Regen1, Janina Kuschmann1, Florian Wanke1, Laureen A Gabriel1, Judith Hauptmann1, Werner Müller2, Emmanuel Pinteaux2, Florian C Kurschus1 and Ari Waisman *,1 1Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany 2Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK *Corresponding author. Tel: +49 6131 17 9360; E-mail: [email protected] *Corresponding author. Tel: +49 6131 17 9129; E-mail: [email protected] The EMBO Journal (2017)36:102-115https://doi.org/10.15252/embj.201694615 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Interleukin-1 (IL-1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which IL-1 is involved in the generation of pathogenic T cells and in disease development remains largely unknown. We found that following EAE induction, pertussis toxin administration leads to IL-1 receptor type 1 (IL-1R1)-dependent IL-1β expression by myeloid cells in the draining lymph nodes. This myeloid-derived IL-1β did not vitally contribute to the generation and plasticity of Th17 cells, but rather promoted the expansion of a GM-CSF+ Th17 cell subset, thereby enhancing its encephalitogenic potential. Lack of expansion of GM-CSF-producing Th17 cells led to ameliorated disease in mice deficient for IL-1R1 specifically in T cells. Importantly, pathogenicity of IL-1R1-deficient T cells was fully restored by IL-23 polarization and expansion in vitro. Therefore, our data demonstrate that IL-1 functions as a mitogenic mediator of encephalitogenic Th17 cells rather than qualitative inducer of their generation. Synopsis Induction of autoimmune neuroinflammation requires expansion of self-antigen-specific CD4 T cells in an IL-1-dependent manner, which can be substituted by IL-23 stimulation. Pertussis toxin promotes IL-1β expression by myeloid cells. Th17 cells express higher levels of IL-1R1 compared to other CD4 T cells. IL-1 signaling results in the expansion of autoreactive Th17 cells. IL-23 can substitute the lack of IL-1 signaling in CD4 T cells. Introduction Differentiated CD4 T cells play an essential role in defense against pathogens and are culprits in autoimmune disorders. Their implication among other diseases is well documented in multiple sclerosis (MS), an autoimmune dysfunction affecting the human central nervous system (CNS), and in its animal model experimental autoimmune encephalomyelitis (EAE) (Croxford et al, 2011). CD4 T-cell involvement in CNS autoimmunity was initially associated with T helper cells producing IFNγ (Th1 cells) (Ando et al, 1989). However, with the discovery of cells expressing IL-17 (Th17 cells) (Harrington et al, 2005; Park et al, 2005), this paradigm was challenged. Th17 cells represent a heterogeneous population of cells expressing not only IL-17 but also additional cytokines, depending on the conditions available during their differentiation. Thus, Th17 cells were found to co-express IFNγ (Ivanov et al, 2006), GM-CSF (Codarri et al, 2011; El-Behi et al, 2011), IL-21 (Korn et al, 2007), IL-22 (Kreymborg et al, 2007), and even the suppressive cytokine IL-10 (McGeachy et al, 2007; Ghoreschi et al, 2010). Moreover, while the impact of IL-17 itself for EAE pathogenesis is still controversial (Haak et al, 2009; Ishigame et al, 2009), GM-CSF is indispensable for disease development (Codarri et al, 2011; El-Behi et al, 2011). In line with that, several recent reports indicated GM-CSF as an essential effector molecule also in MS (Hartmann et al, 2014; Noster et al, 2014; Li et al, 2015). Th17 cell differentiation from naïve T cells can be initiated by TGFβ, the cytokine that is shared with the T regulatory (Treg) cell developmental program. However, Th17 cell commitment occurs only after subsequent inhibition of the Treg-specific transcription factor FoxP3 by IL-6 (Bettelli et al, 2006; Veldhoen et al, 2006) or IL-21 (Korn et al, 2007). Th17 cells generated only in the presence of TGFβ1 and IL-6 cannot transmit EAE (McGeachy et al, 2007) and require additional exposure to IL-23 to gain pathogenic characteristics (Lee et al, 2012). Another pathway of Th17 cell development is independent of TGFβ and driven by a combination of IL-6, IL-23, and IL-1β (Chung et al, 2009; Ghoreschi et al, 2010). Cells generated under these conditions express high levels of GM-CSF (El-Behi et al, 2011) and are highly pathogenic in EAE. Of note, the individual ablation of each aforementioned cytokine-mediated signaling results in complete resistance of mice to EAE induction (Eugster et al, 1998; Cua et al, 2003; Matsuki et al, 2006; Sutton et al, 2006). Whereas deletion of IL-6 prevented Th17 cell differentiation (Korn et al, 2007), IL-23 showed a greater impact on expansion and stabilization of the Th17 cell phenotype (Langrish et al, 2005; Veldhoen et al, 2006). The contribution of IL-1 signaling to the pathogenicity of T cells is still controversial with evidences supporting a role in both de novo generation and expansion of Th17 cells (Sutton et al, 2006; Chung et al, 2009; El-Behi et al, 2011). The mechanism by which IL-1 mediates pathogenicity in vivo could not yet be fully addressed, mainly due to the lack of suitable genetic tools, namely the conditional knockout of the IL-1 receptor. There is only one known signaling receptor—IL-1 receptor type 1 (IL-1R1)—that is, however, broadly expressed by many cell types of immune and non-immune origin (Boraschi & Tagliabue, 2013). The induction of active EAE is achieved by the immunization with myelin oligodendrocyte glycoprotein (MOG), emulsified in complete Freund's adjuvant (CFA) and injections of pertussis toxin (PTx) (Mendel et al, 1995). PTx is commonly used in many autoimmune models to enhance disease susceptibility; however, its mode of action is not fully understood. In EAE, PTx application is associated with transient Treg cell suppression (Cassan et al, 2006; Chen et al, 2006), promotion of Th17 cell development (Chen et al, 2007), and contribution to blood–brain barrier disruption (Millward et al, 2007). On the other hand, PTx is also able to block leukocyte migration (Su et al, 2001), demonstrating its complex and diverse involvement in disease progression. Despite these proposed effects of PTx, many of them most likely are secondary events with largely unknown key intermediate factor(s). Importantly, PTx also was shown to stimulate expression and inflammasome-mediated maturation of IL-1β in myeloid cells (Dumas et al, 2014), suggesting that IL-1β could be a potential candidate molecule through which PTx would, at least partially, promote EAE development. In the present study, we aimed to investigate the requirement of IL-1 signaling for T-cell-mediated autoimmunity. By using novel models of conditional IL-1R1 deletion, we demonstrate that PTx treatment stimulates recruitment and expansion of myeloid cells expressing IL-1β in the draining lymph nodes (dLN), in an IL-1R1-dependent manner. This myeloid cell-derived IL-1 stimulated expansion of GM-CSF+ Th17 cells. The latter cells also developed in the absence of IL-1 signaling but did not proliferate properly and therefore could not cause severe EAE. However, isolation and expansion of T cells devoid of IL-1 signaling in the presence of IL-23 resulted in recovery of pathogenicity. Thus, we could show that IL-1 signaling in CD4 T cells is critical for their expansion, but not for differentiation to encephalitogenic Th17 cells. Results PTx administration promotes IL-1β expression by myeloid cells during the induction phase of EAE in an IL-1R1-dependent manner Recently, we generated a new mouse line that allows for the conditional deletion of the IL-1 receptor type 1 (Abdulaal et al, 2016), the obligatory receptor for IL-1α- and IL-1β-mediated signaling. By deleting the receptor in the germ line, using CMV-Cre, we obtained mice that globally lack IL-1R1 (hereafter termed IL-1R1−/−). These mice differ from previously reported full knockout mice by the deletion of exon 5, which results in inactivation of all known isoforms for IL-1R1 (Glaccum et al, 1997; Labow et al, 1997; Qian et al, 2012). As a result of complete inactivation of IL-1 signaling, IL-1R1−/− mice displayed impaired development of CD11b+ cells in secondary lymphoid organs under steady state conditions, including myeloid cells producing IL-1β, as compared to wild-type (WT) controls (Fig 1A–C). Figure 1. Pertussis toxin induces IL-1β-expressing myeloid cells after MOG/CFA immunization dependent on IL-1R1 signaling Frequencies and total numbers (mean + SEM) of CD11b+ myeloid cells isolated from the spleen and peripheral LN of naïve non-immunized mice. Total numbers (mean + SEM) of CD11b+ cells expressing IL-1β shown in (A) and restimulated with indicated stimuli for 4 h. Quantification (mean + SEM) of myeloid cell populations expressing IL-1β isolated from LN and restimulated with GM-CSF shown in (B). Frequencies and total numbers (mean + SEM) of CD11b+ myeloid cells isolated from the spleen of immunized mice. Analysis of IL-1β expression by CD11b+ cells isolated from dLN of mice treated as in (D). Data are representative FACS histogram overlays, gated on VD−CD11b+ cells with mean frequencies per group. Quantification (mean + SEM) of IL-1β expression by CD11b+ cells shown in (E). Analysis of IL-1β-expressing CD11b+ cells shown in (E). Data are representative FACS plots, gated on VD−CD11b+IL-1β+ cells with mean frequencies per group. Quantification (mean + SEM) of myeloid cell populations expressing IL-1β shown in (G). Data information: Neutrophils were defined as CD11b+Ly6G+Ly6Clow cells, monocytes/macrophages as CD11b+Ly6G−Ly6Chigh cells. Cells were isolated (A–C) from naïve non-immunized mice n = 5 of each genotype; (D–H) at day 7 after immunization from n = 4 WT PBS-, n = 3 IL-1R1−/− PBS-, n = 4 WT PTx-, n = 3 IL-1R1−/− PTx MOG/CFA-immunized mice. Cells (A, C, D–H) were restimulated with 20 ng/ml GM-CSF and (B) with GM-CSF or 500 ng/ml LPS in the presence of monensin for 4 h. *P < 0.05, **P < 0.01, N.S., not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results. Download figure Download PowerPoint Experimental autoimmune encephalomyelitis can be induced in mice by immunization with the MOG peptide p35-55 emulsified in CFA, along with PTx injections. To study the role of IL-1 signaling in EAE induction, we immunized mice with MOG/CFA with or without addition of PTx. We found dramatically reduced frequencies and numbers of CD11b+ myeloid cells in the spleen of IL-1R1-deficient animals compared to wild-type controls (Fig 1D). These experiments suggest that IL-1 signaling might be involved in the recruitment of myeloid cells to the secondary lymphoid organs and their subsequent expansion after immunization. To investigate whether mobilized myeloid cells not only respond to IL-1 but also enhance the immune response by producing this cytokine, we measured the expression of IL-1β upon immunization. Following stimulation with GM-CSF, LPS, or PMA/ionomycin to trigger ex vivo isolated cells, we found that the vast majority of IL-1β originated from CD11b+ cells (Fig EV1). Moreover, we noted a robust enhancement of IL-1β expression by myeloid cells when WT animals were additionally treated with PTx, an effect that was completely absent in IL-1R1-deficient animals (Figs 1E and F, and EV1). Further analysis of the myeloid cell populations revealed that treatment of the mice with PTx resulted in increased frequencies of neutrophils and monocytes/macrophages among the cells expressing IL-1β in the WT group, whereas it had a very limited effect on the same cell populations in IL-1R1−/− mice (Fig 1G and H). In contrast to IL-1β, the expression of IL-1α in myeloid cells was not affected by PTx treatment (Fig EV2). However, in line with the IL-1β data, IL-1α-expressing CD11b+ cells were dramatically reduced in mice deficient for IL-1R1 (Fig EV2). Click here to expand this figure. Figure EV1. Myeloid cells are the main source of IL-1β upon MOG/CFA/PTx immunization A–C. Analysis of IL-1β expression by cells isolated from the dLN and stimulated with GM-CSF (A), LPS (B), and PMA/ionomycin (C). Data are representative FACS plots gated on VD− cells with mean frequencies per group. Data information: Cells (A–C) were isolated at day 7 after immunization and stimulated in the presence of monensin with indicated stimuli for 4 h. Data consist of n = 4 WT PBS-, n = 3 IL-1R1−/− PBS-, n = 4 WT PTx-, n = 3 IL-1R1−/− PTx MOG/CFA-immunized mice. Experiments were performed twice with similar results. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Myeloid cells are the main source of IL-1α upon MOG/CFA/PTx immunization Analysis of IL-1α expression by cells isolated from the spleen and stimulated with GM-CSF. Data are representative FACS plots, gated on VD− cells with mean frequencies per group. Frequencies (mean + SEM) of IL-1α expression by CD11b+ cells shown in (A). Data information: Cells (A, B) were isolated at day 7 after immunization and were stimulated with 20 ng/ml GM-CSF for 4 h in the presence of monensin. Data consist of n = 4 WT PBS-, n = 3 IL-1R1−/− PBS-, n = 4 WT PTx-, and n = 3 IL-1R1−/− PTx MOG/CFA-immunized mice. *P < 0.05, N.S., not significant; two-tailed unpaired t-test. Experiments were performed twice with similar results. Download figure Download PowerPoint Collectively, these data show that PTx activates myeloid cells to produce IL-1β in an IL-1R1-dependent manner after MOG/CFA immunization. In addition, our data suggest that IL-1β production by myeloid cells is dependent on an autocrine stimulatory feedback loop. Mouse and human Th17 cells express high levels of IL-1R1 Development of EAE requires the generation of pathogenic CD4 T cells that initiate a coordinated immune attack of the CNS. To study the role of IL-1 signaling in the development of a T-cell-mediated immune response, we investigated the expression of IL-1R1 by different populations of CD4 T cells following immunization with MOG/CFA/PTx (Appendix Fig S1). We found that Th17 cells expressed the highest levels of IL-1R1 compared to conventional Th1 or GM-CSF-producing T cells in both the dLN and the inflamed CNS of EAE-diseased mice (Fig 2A and C). We also noted that FoxP3+ Treg cells hardly expressed any IL-1R1 (Fig 2A and C). Importantly, IL-17A-expressing cells regardless of their IFNγ and/or GM-CSF co-expression equally displayed high levels of IL-1R1 (Fig 2B and D). Figure 2. IL-1R1 expression by murine and human Th17 cells A–D. Analysis of IL-1R1 expression by different populations of murine (A) CD4 T cells and (B) Th17 cells isolated from the dLN 9 days after immunization, and (C) CD4 T cells and (D) Th17 cells isolated from the CNS at the peak of EAE. E, F. Analysis of IL-1R1 expression by different populations of human (E) CD4 T cells and (F) Th17 cells isolated from the peripheral blood. Data information: Data (A, C) are representative FACS histogram overlay of VD−TCRβ+CD4+ cells gated on IL-17A+, IL-17A−IFNγ+ (IFNγ+), IL-17A−GM-CSF+ (GM-CSF+), or IL-17A−FoxP3+ (FoxP3+) cells (also see Appendix Fig S1) and quantifications shown as bar diagram (mean + SEM). Data (E) are FACS plots of cells gated on CD14−CD3+CD4+ cells with mean frequencies per group and quantifications shown as bar diagram (mean + SEM). Data (B, D, F) are FACS plots of Th17 cells and histograms of IL-1R1 staining of indicated Th17-cell subpopulations with mean fluorescent intensity (MFI) ± SEM or mean frequencies per group. Cells (A–D) were restimulated in the presence of MOG for 6 h. Data consist of n = 4 wild-type mice immunized with MOG/CFA/PTx. Cells (E, F) were restimulated with PMA/ionomycin for 4 h. Data consist of PBMC isolated from n = 4 healthy individuals. *P < 0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired t-test. Experiments were performed twice with similar results. Download figure Download PowerPoint Also in humans, CD4 T-cell development was shown to be dependent on IL-1 signaling (Acosta-Rodriguez et al, 2007; Zielinski et al, 2012). Although mouse models represent useful tools in investigating human pathologies, possible differences in cell biology can result in misleading translation of the findings to the clinic. In line with our mouse data, we found that a large proportion of human Th17 cells also expressed IL-1R1, but very few Th1- and GM-CSF-positive cells do so (Fig 2E). When we further analyzed the different subpopulations of IL-17A-producing human CD4 T cells, we found that all of them, regardless of co-expression of IFNγ and/or GM-CSF, expressed similar levels of IL-1R1 (Fig 2E and F). We conclude that both mouse and human subpopulations of Th17 cells, including the most autoaggressive GM-CSF+ Th17 cells, can potentially respond to IL-1 and therefore could be dependent on IL-1 signaling. PTx enhances the development of MOG-specific GM-CSF+ Th17 cells in an IL-1R1-dependent manner To investigate the role of PTx-induced IL-1β expression on the development of autoreactive T cells, we compared cytokine production by MOG-specific CD4 T cells generated after MOG/CFA immunization of mice with or without PTx injections. To visualize antigen-specific T cells, we re-activated spleen- and dLN-derived lymphocytes with MOG peptide for 6 h and addressed the expression of CD40 ligand (CD40L) together with the cytokine staining. CD40L is transiently expressed by T cells after their activation and can be used to identify antigen-specific T cells (Chattopadhyay et al, 2005; Frentsch et al, 2005). In WT mice, addition of PTx to the immunization protocol doubled numbers of MOG-reactive Th17 cells (Fig 3A and B). This effect of PTx was strongly dependent on IL-1R1, since we did not observe it in mice lacking the IL-1 receptor. Instead, IL-1R1−/− mice showed a dramatic reduction of antigen-specific Th17 cells compared to WT animals, regardless of the addition of PTx (Fig 3A and B). As a negative control, we restimulated cells with the non-relevant OVA peptide and could not detect OVA-specific cells neither in WT nor in IL-1R1-deficient animals (Fig EV3). Figure 3. GM-CSF-expressing Th17 cells are expanded by PTx in response to IL-1 signaling A. Analysis of cytokine expression by CD4 T cells isolated from the spleen. Data are representative FACS plots, gated on VD−TCRβ+CD4+CD44+ cells (upper row) with mean frequencies among CD4 T cells per group ± SEM and (lower row) with mean frequencies per group. B. Frequencies and total numbers (mean + SEM) of MOG-specific Th17 cells isolated from the dLN. C, D. Frequencies and total numbers (mean + SEM) of MOG-specific GM-CSF+ Th17 cells isolated from the spleen (C) and dLN (D). Data information: Cells (A–D) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n = 5 WT PBS-, n = 4 WT PTx-, n = 4 IL-1R1−/− PBS-, n = 4 IL-1R1−/− PTx MOG/CFA-immunized mice. *P < 0.05, **P < 0.01, N.S., not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. IL-1R1 deletion impairs MOG-specific Th17 cell expansion Analysis of IL-17A expression by CD4 T cells isolated from the spleen and restimulated with MOG or OVA for 6 h. Total cell numbers of antigen-specific Th17 cells isolated from the spleen shown in (A). Data information: Cells (A, B) were isolated at day 9 after MOG/CFA/PTx immunization of n = 4 mice of each genotype. Data are (A) representative FACS plots, gated on VD−TCRβ+CD4+CD44+ cells with mean frequencies among CD4 T cells per group ± SEM and (B) bar diagram (mean + SEM). *P < 0.05, N.S., not significant; two-tailed unpaired t-test. Download figure Download PowerPoint Importantly, the stimulatory effect of PTx observed in WT mice was not restricted to Th17 cells but was also seen in CD4 T cells expressing IFNγ, GM-CSF, or both (Fig EV4). These data are in agreement with our findings that IL-1R1−/− mice displayed very limited numbers of such cells regardless of PTx administration (Fig EV4). Previously, our group and others have shown that in the context of EAE, IFNγ-, and GM-CSF-positive cells are mainly (or even exclusively) originating from Th17 cells (Kurschus et al, 2010; Hirota et al, 2011; Brucklacher-Waldert et al, 2016). Taken together, we conclude that deficiency in IL-1 signaling (either by IL-1R1 deletion or absence of PTx during immunization) results in reduced Th17 cells and, as expected, their IFNγ- and GM-CSF-expressing progenies, during priming of EAE. Click here to expand this figure. Figure EV4. IL-17A-related cytokine expression by CD4 T cells is dependent on IL-1R1 signaling A–G. Analysis of cytokine expression by CD4 T cells isolated from the (A, B, D, F) spleen and (C, E, G) dLN (depicted in Fig 3). Frequencies and total numbers of MOG-specific (B, C) GM-CSF+IFNγ+ cells, (D, E) GM-CSF+ cells, and (F, G) IFNγ+ cells. Data (A) are representative FACS plots, gated on VD−TCRβ+CD4+CD44+CD40L+ cells with mean frequencies among CD44+CD40L+ cells per group, and (B–G) bar diagram (mean + SEM). Data information: Cells (A–G) were isolated at day 9 after immunization and restimulated in the presence of MOG for 6 h. Data consist of n = 5 WT PBS-, n = 4 WT PTx-, n = 4 IL-1R1−/− PBS-, n = 4 IL-1R1−/− PTx MOG/CFA-immunized mice. *P < 0.05, **P < 0.01, ***P < 0.001, N.S., not significant; two-tailed unpaired t-test. Experiments were performed at least twice with similar results. Download figure Download PowerPoint As mentioned above, following EAE induction, Th17 cells go through a process of differentiation that results in the co-expression of IFNγ and GM-CSF, which are critical for the pathogenicity of CD4 T cells (Kurschus et al, 2010; Codarri et al, 2011; El-Behi et al, 2011; Hirota et al, 2011). Therefore, we further analyzed antigen-specific IL-17A-producing cells for the production of IFNγ and GM-CSF. In WT mice, we found that frequencies and numbers of MOG-specific Th17 cells co-expressing GM-CSF were significantly higher when mice were injected with PTx compared to PBS-treated animals (Fig 3A, C and D). The vast majority of IFNγ-positive Th17 cells also expressed GM-CSF, while IFNγ single positive Th17 cells represented only a minor subpopulation regardless of the PTx administration (Fig 3A). Importantly, the stimulatory effect of PTx was not observed in mice deficient for IL-1R1, in which autoreactive IL-17A+ cells displayed impaired co-expression of GM-CSF in both the spleen and dLN (Fig 3C and D). Collectively, these findings suggest that PTx enhances the generation of pathogenic Th17 cells and particularly its GM-CSF-positive subset, by promoting their expansion via IL-1 signaling. Regulatory T cells are generated early after EAE induction (O'Connor & Anderton, 2008; Yogev et al, 2012), and their development was shown to be transiently suppressed by PTx (Cassan et al, 2006; Chen et al, 2006). However, we did not observe any effects of PTx administration or IL-1R1 deletion on the numbers of FoxP3+ CD4 T cells in dLN and spleen when mice were analyzed 9 days after immunization (Appendix Fig S2A–C). Moreover, expression levels of Treg co-stimulatory molecules GITR, ICOS, and CTLA-4 were also unchanged in all groups of animals (Appendix Fig S2D and E). We conclude that neither IL-1 signaling, nor PTx administration is critically influencing Treg cell development following EAE induction. IL-1 signaling in T cells is essential for expansion of antigen-specific GM-CSF+ Th17 cells To investigate the exact role of IL-1 in the development of autoreactive T cells and their pathogenicity, we specifically deleted the IL-1 receptor type 1 in αβ-TCR+ T cells by crossing conditional IL-1R1fl/fl mice to CD4-Cre mice, resulting in IL-1R1∆T mice. These mice developed normally and did not display abnormalities in the immune system when kept under SPF conditions. However, CD4 T cells isolated from mutant mice failed to proliferate in response to IL-1β administration in vitro (Mufazalov et al, 2016). Previously, it was reported that IL-1R1 fully deficient mice show an impaired CD4 T-cell-mediated antigen response and that these mice are resistant to induction of EAE (Matsuki et al, 2006; Sutton et al, 2006; Chung et al, 2009). To test whether EAE resistance specifically stems from the lack of T-cell responses to IL-1, we immunized WT, IL-1R1−/−, and IL-1R1∆T mice with MOG/CFA along with two injections of PTx and investigated priming of CD4 T cells 9 days postimmunization. As expected, we