Interleukin-27 Gene Therapy Prevents the Development of Autoimmune Encephalomyelitis but Fails to Attenuate Established Inflammation due to the Expansion of CD11b+Gr-1+ Myeloid Cells

Interleukin-27 (IL-27) and its subunit P28 (also known as IL-30) have been shown to inhibit autoimmunity and have been suggested as potential immunotherapeutic for autoimmune diseases such as multiple sclerosis (MS). However, the potential of IL-27 and IL-30 as immunotherapeutic, and their mechanisms of action have not been fully understood. In this study, we evaluated the efficacy of adeno-associated viral vector (AAV)-delivered IL-27 (AAV-IL-27) and IL-30 (AAV-IL-30) in a murine model of MS. We found that one single administration of AAV-IL-27, but not AAV-IL-30 completely blocked the development of experimental autoimmune encephalomyelitis (EAE). AAV-IL-27 administration reduced the frequencies of Th17, Treg, and GM-CSF-producing CD4+ T cells and induced T cell expression of IFN-γ, IL-10, and PD-L1. However, experiments involving IL-10-deficient mice and PD-1 blockade revealed that AAV-IL-27-induced IL-10 and PD-L1 expression were not required for the prevention of EAE development. Surprisingly, neither AAV-IL-27 nor AAV-IL-30 treatment inhibited EAE development and Th17 responses when given at disease onset. We found that mice with established EAE had significant expansion of CD11b+Gr-1+ cells, and AAV-IL-27 treatment further expanded these cells and induced their expression of Th17-promoting cytokines such as IL-6. Adoptive transfer of AAV-IL-27-expanded CD11b+Gr-1+ cells enhanced EAE development. Thus, expansion of CD11b+Gr-1+ cells provides an explanation for the resistance to IL-27 therapy in mice with established disease.

Interleukin-27 (IL-27) and its subunit P28 (also known as IL-30) have been shown to inhibit autoimmunity and have been suggested as potential immunotherapeutic for autoimmune diseases such as multiple sclerosis (MS). However, the potential of IL-27 and IL-30 as immunotherapeutic, and their mechanisms of action have not been fully understood. In this study, we evaluated the efficacy of adeno-associated viral vector (AAV)-delivered IL-27 (AAV-IL-27) and IL-30 (AAV-IL-30) in a murine model of MS. We found that one single administration of AAV-IL-27, but not AAV-IL-30 completely blocked the development of experimental autoimmune encephalomyelitis (EAE). AAV-IL-27 administration reduced the frequencies of Th17, Treg, and GM-CSF-producing CD4 + T cells and induced T cell expression of IFN-γ, IL-10, and PD-L1. However, experiments involving IL-10-deficient mice and PD-1 blockade revealed that AAV-IL-27-induced IL-10 and PD-L1 expression were not required for the prevention of EAE development. Surprisingly, neither AAV-IL-27 nor AAV-IL-30 treatment inhibited EAE development and Th17 responses when given at disease onset. We found that mice with established EAE had significant expansion of CD11b + Gr-1 + cells, and AAV-IL-27 treatment further expanded these cells and induced their expression of Th17-promoting cytokines such as IL-6. Adoptive transfer of AAV-IL-27-expanded CD11b + Gr-1 + cells enhanced EAE development. Thus, expansion of CD11b + Gr-1 + cells provides an explanation for the resistance to IL-27 therapy in mice with established disease.
Indeed, previous studies (9,13) have revealed that IL-27 delivered systemically can inhibit the development of experimental autoimmune encephalomyelitis (EAE) in mice, an experimental model of MS. However, systemic injection of IL-27 is costly, and it is also difficult to maintain an effective concentration in the circulation. In this context, gene therapy could serve as an effective alternative approach. For instance, IL-30 gene therapy has been shown to efficiently inhibit autoimmune inflammation in the central nervous system (CNS) and eye (16), and lentiviral IL-27 gene delivery to the CNS inhibits neuroinflammation (17). Adeno-associated viral vectors (AAVs) are highly efficient delivery agents for gene therapy (18). AAV vectors can efficiently transfer genes of interest to a broad range of mammalian cell types leading to high levels of stable and long-term expression after a single application (19). AAV vectors are also known to have low immunogenicity and have been used in human clinical trials (20)(21)(22). In this study, we have evaluated the therapeutic efficacy of AAV-delivered IL-27 (AAV-IL-27) and IL-30 (AAV-IL-30) in T cell-mediated autoimmune encephalomyelitis, where the inflammation in the CNS is considered to be mediated mainly by Th17/Th1 responses and T cells producing GM-CSF (23,24). We found that one single administration of AAV-IL-27, but not AAV-IL-30 completely prevented EAE development. Experiments involving IL-10-deficient mice and PD-1 blockade revealed that AAV-IL-27-induced IL-10 and PD-L1 expression were not required for the inhibition of EAE development. However, neither AAV-IL-27 nor AAV-IL-30 treatment inhibited EAE development and Th17 responses when given at disease onset. We found that mice with established EAE had significant expansion of CD11b + Gr-1 + myeloid cells, and AAV-IL-27 treatment further expanded these cells and induced their expression of multiple cytokines including Th17-promoting cytokines such as IL-6 and IL-23. Adoptive transfer of AAV-IL-27-expanded CD11b + Gr-1 + cells enhanced EAE development. Thus, systemic delivery of IL-27 can efficiently prevent EAE development and the priming of Th17 responses. However, the therapeutic potential of IL-27 is limited by its failure in inhibiting ongoing EAE, and shutting down established Th17 responses, presumably due to the expansion of CD11b + Gr-1 + myeloid cells.

MaTerials anD MeThODs
Mice C57BL/6, C57BL/6 mice with targeted mutation of the IL-27Rα (IL-27Rα −/− ) and IL-10 (IL-10 −/− ) genes were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). 2D2 TCR transgenic mice (25) were described before (26,27). All mice were maintained in the animal facilities of The Ohio State University, and the studies were approved by the Institutional Animal Care and Use Committee.
induction and assessment of eae C57BL6, IL-10 −/− , and 2D2 mice of 8-12 weeks of age were immunized subcutaneously with 200 µg MOG 35-55 emulsified in PBS:CFA (1:1) in a total volume of 100 µL. MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from Genemed Synthesis, Inc. (South San Francisco, CA, USA). The purity of the peptide was greater than 90%. Mice also received 150 ng of pertussis toxin (List Biological, Campbell, CA, USA) in 200 µL PBS via the tail vein immediately after the immunization and again 48 h later. The mice were observed every day for the development of EAE symptoms using parameters as we described before (26,27).

Production of aaV Viruses and Mice Treatment
Adeno-associated viral vector-IL-27, AAV-IL-30, and AAV-ctrl viruses were produced as we previously described (28). Briefly, IL-27 or IL-30 cDNA were inserted into an AAV carrier vector under the control of the CMV-chicken beta-actin hybrid promoter (29,30). The IL-27 or IL-30 carrier AAV vector was compacted with a helper vector in 293K cells into the AAV serotype 8 (AAV8), which could achieve high expression in muscles (31,32). AAV viruses were injected into mice intramuscularly (i.m.) using a dose of 2 × 10 11 DRP/mouse diluted in 50 µL PBS.

isolation of Mononuclear cells From spinal cords
Spinal cord tissues from AAV-IL-27, AAV-ctrl virus-treated or -untreated mice with EAE were removed and cut into about 2-mm pieces and incubated in 10 mM Hepes/NaOH buffer containing 1 mg/mL of collagenase IV (Sigma, St. Louis, MO, USA) for 1 h at 37°C. Then, the tissues were dispersed with syringe, filtered through a 100-mm wire mesh, and centrifuged at 2,000 rpm for 5 min at 4°C. After centrifugation, tissue pallets were resuspended in 15 mL 30% Percoll (Pharmacia, Uppsala, Sweden), then centrifuged against 70% Percoll in a 50-mL tube for 15 min. The cell monolayer at the 30-70% Percoll interface was collected and washed once for further staining and flow cytometry analyses. Procedures for cell surface marker staining and intracellular cytokine staining were the same as we described (26,27). Briefly, for staining of cell surface markers, mononuclear cells from spleens, lymph nodes, and CNS were stained with various antibodies in staining buffer (PBS with 1% FCS) and incubated on ice for 30 min. After washing with staining buffer, cells were fixed in 1% paraformaldehyde in PBS. For intracellular cytokine staining, cells were stimulated in culture medium for 4 h with 100 ng/mL of phorbol 12-myristate 13-acetate and 500 ng/mL of ionomycin in the presence of Golgi stop (1:1,500; BD Biosciences). Viable cells were then fixed in IC fixation buffer (eBioscience), permeabilized with 1× permeabilization buffer (eBiosciences), and stained with respective antibodies. Foxp3 staining was performed according to the manufacturer's protocol (BD Biosciences). Cells were collected on a FACSCalibur flow cytometer, and data were analyzed using the FlowJo software (Tree Star, Inc., OR, USA).

antibodies and Flow cytometry
sorting of cD11b + gr-1 + cells and adoptive Transfer into Mice With established eae Spleen mononuclear cells from AAV-IL-27 or AAV-ctrl virustreated mice (with or without EAE) were stained for CD11b and Gr-1, the CD11b + Gr-1 + cells were then sorted using the Moflo XDP sorter (Beckman Coulter, Indianapolis, IN, USA). To treat mice with EAE using CD11b + Gr-1 + myeloid cells, we first established EAE in C57BL6 mice, on day 10 post-immunization, mice were treated with AAV-IL-27 or AAV-ctrl virus as described above. Fourteen days after AAV treatment, mice were sacrificed and CD11b + Gr-1 + myeloid cells were sorted from spleens and were injected i.v. into mice with established EAE (1 million cells/ per mouse; day 10 post EAE induction). The mice were observed for EAE development.

resUlTs systemic Delivery of il-27 by aaV Virus inhibits Th17 responses and Prevents eae Development
To determine if IL-27 or IL-30 can be used as a potential therapeutic for autoimmune diseases, we generated recombinant adenoassociated virus that express IL-27 (AAV-IL-27) or IL-30 (AAV-IL-30) and the control AAV virus (AAV-ctrl). Intramuscular injection (i.m.) of 2 × 10 11 DRP/mouse of AAV-IL-27 or AAV-IL-30 achieved high and stable IL-27 ( Figure 1A) or IL-30 ( Figure 1B) production in the peripheral blood of mice. AAV-IL-27 treatment significantly enhanced Th1 response and slightly induced T cell production of IL-10, while reduced the frequencies of Th17 and Treg cells in spleens ( Figure 1C). By contrast, AAV-IL-30 treatment slightly inhibited Th1 response but failed to affect the frequencies of Th17/Treg cells and T cell production of IL-10 ( Figure 1D).
To determine if AAV-delivered IL-27 or IL-30 could block EAE development, we injected AAV-IL-27, AAV-IL-30, or AAV-ctrl virus into C57BL6 mice, 1 week later mice were immunized with MOG35-55/CFA and pertussis toxin. In AAV-ctrl virus-treated mice, EAE symptoms developed, with first symptoms showed up on day 10, while disease progressed to peak around days 14-17, then the EAE symptoms went down but maintained at a lower level for a long time (Figures 2A,B). While a single injection of AAV-IL-27 completely prevented EAE development in C57BL6 mice (Figure 2A), a single dose of AAV-IL-30 only slightly inhibited EAE development ( Figure 2B). AAV-IL-27 treatment failed to prevent EAE in IL-27Rα −/− mice, suggesting that AAV-IL-27 acts through IL-27 receptor ( Figure 2C). 2D2 TCR transgenic mice develop progressive EAE symptoms upon immunization, presumably due to the activation of overwhelming numbers of myelin-specific T cells. We therefore tested if EAE in 2D2 mice could be prevented by AAV-IL-27 treatment. As shown in Figure 2D, AAV-IL-27 administration slightly delayed the onset of EAE symptoms, but significantly inhibited the EAE symptoms in 2D2 mice.
To determine if AAV-IL-27 prevented EAE development by altering T cell responses, we analyzed T cell subsets in the draining lymph nodes (DLNs) and spleens from AAV-IL-27-treated mice and controls. As shown in Figure 3, we found that the CD4 + T cells from the immune lymph nodes ( Figure 3A) and Since IL-27 was shown to induce T cell expression of PD-L1, which contributed to T cell tolerance in the EAE model (10), we tested if AAV-IL-27 induced T cell tolerance through induction of PD-L1. As shown in Figure 4B, we found that treatment with AAV-IL-27, but not AAV-Ctrl virus indeed induced significant expression of PD-L1 in T cells. To determine if PD-L1-PD-1 interaction among T cells mediated their tolerance, C57BL6 mice were first treated with AAV-IL-27 or AAV-ctrl virus followed by EAE induction 1 week later. On days 5,9,13, and 17 after EAE induction, mice receiving AAV-IL-27 treatment were also treated with 300 μg/mouse of anti-PD-1 or an isotype-matched control antibody i.p. As shown in Figure 4C, while mice treated with AAV-ctrl virus and control antibody exhibited EAE symptoms by day 10 and reached peak disease by day 17, mice treated with AAV-ctrl virus and anti-PD-1 developed more severe EAE, consistent with the known functions of PD-1 blockade in EAE development (34). However, mice treated with AAV-IL-27 + ctrl antibody or AAV-IL-27 + anti-PD-1 showed no EAE symptoms ( Figure 4C). Thus, blockade of PD-L1-PD-1 interaction failed to reverse T cell tolerance induced by AAV-IL-27 treatment.

aaV-il-27 Treatment Does not inhibit established Th17 responses and eae
To determine if AAV-IL-27 treatment could reverse ongoing inflammation in the CNS, C57BL6 mice were immunized with MOG peptide/CFA and pertussis toxin. Ten days after immunization, when the first symptoms of EAE appeared, mice were treated with AAV-IL-27 or AAV-Ctrl virus i.m. As shown in Figure 5A, AAV-IL-27 treatment at day 10 after EAE induction failed to inhibit EAE development. Similarly, we found that treatment of mice on day 10 after EAE induction with AAV-IL-30 also had no effect on EAE development (Figure 5B). One potential explanation for failure of inhibiting EAE development could be due to lack of IL-27 receptor expression in the CNS-infiltrating CD4 + T cells. However, we found high levels of IL-27Rα expression in the CNS-infiltrating CD4 + T cells ( Figure 5C). Moreover, we found that AAV-IL-27 treatment induced PD-L1 expression in CNS-infiltrating CD4 + T cells ( Figure 5D) and enhanced Th1 responses without significantly affecting Th17 responses in the CNS (Figure 5E). AAV-IL-27-treatment also significantly inhibited Treg subset without significantly affecting Tr1 subset in the CNS (Figure 5F). GM-CSF-producing CD4 + T cells were found to be increased in the CNS of AAV-IL-27 treated mice ( Figure 5F). Strikingly, we found that AAV-IL-27 treatment of mice with ongoing EAE upregulated many cytokine genes including GM-CSF, IL-17, IL-10, IFN-γ, IL-6, IL-1β, and TNF-α in the CNS (Figure 5G).
aaV-Mediated Delivery of il-27 induces the expansion of cD11b + gr1 + Myeloid cells Significant induction of cytokines such as IL-6, IL-1β, and TNF-α in the CNS suggests that AAV-IL-27 treatment may have significant impacts on myeloid cells. Indeed, through the analysis of the myeloid compartment in the peripheral lymphoid organs and CNS, we found that CD11b + Gr1 + myeloid cells were significantly increased in the spleens and CNS of mice with EAE, and AAV-IL-27 treatment further expanded those cells ( Figure 6A). The impact of AAV-IL-27 on this population of cells was dramatic, as in the spleen, CD11b + Gr-1 + myeloid cells expanded about threefold compared to mice with untreated EAE (Figure 6A, right panel). Notably, we did not find expansion of CD11b + Gr-1 + myeloid cells in DLNs ( Figure 6A). The expanded CD11b + Gr-1 + myeloid cells were mainly of the Ly6C low subtype, and subtypes were not significantly different between AAV-IL-27 and AAV-ctrl-treated mice ( Figure 6B). While we observed a major expansion of CD11b + Gr-1 + myeloid cells in mice with EAE that received AAV-IL-27 therapy at disease onset, in the EAE prevention model, AAV-IL-27-treated mice had much lower numbers of CD11b + Gr-1 + myeloid cells compared with AAVctrl-treated mice that developed EAE ( Figure 6C). These results suggest that CD11b + Gr-1 + myeloid cells are mainly associated with disease activity. To determine if AAV-IL-27 therapy directly induce expansion of CD11b + Gr-1 + myeloid cells, we injected AAV-IL-27 or AAV-ctrl virus into naïve C57BL/6 mice and found that AAV-IL-27 treatment could significantly induce expansion of CD11b + Gr-1 + myeloid cells in naïve mice in the absence of EAE ( Figure 6D). These data together suggest that IL-27 alone could induce expansion of CD11b + Gr-1 + myeloid cells, and in the presence of active EAE the expansion of these myeloid cells become more robust. CD11b + Gr-1 + myeloid cells have previously been shown to inhibit or enhance EAE development (35,36). We purified CD11b + Gr-1 + myeloid cells from the spleens of AAV-IL-27 and Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 873 AAV-ctrl virus-treated mice by FACS-based sorting, and analyzed their expression of cytokine genes. As shown in Figure 7A, we found that CD11b + Gr-1 + myeloid cells from AAV-IL-27-treated mice had increased expression of IL-6, IL-17, IL-23, S100A8, A100A9, IL-10, and TNF-α genes. IL-1β expression was decreased compared with myeloid cells from AAV-ctrl treated mice, but remained readily detectable (at 22 cycles by qPCR).
Using flow cytometry analysis, we found that IL-6 protein was readily detectable in CD11b + Gr-1 + myeloid cells from both AAV-IL-27 and AAV-ctrl treated EAE mice (Figure 7B). To test if IL-27-expanded myeloid cells affect EAE development, CD11b + Gr-1 + myeloid cells were FACS-purified from AAV-IL-27 or AAV-ctrl virus-treated mice with EAE and were injected i.v. into mice on day 10 post EAE induction. We found that CD11b + Gr-1 + myeloid cells from AAV-IL-27-treated mice more significantly enhanced EAE development (Figure 7C). Consistent with disease severity, we found that more CD4 + T cells infiltrated into the CNS of mice receiving AAV-IL-27-expanded myeloid cells (Figure 7D).

DiscUssiOn
In this study, we have evaluated the efficacy of AAV-delivered IL-27 (AAV-IL-27) and IL-30 (AAV-IL-30) in a murine model of MS. We found that one single administration of AAV-IL-27 completely prevented autoimmune encephalomyelitis, while significant, but incomplete protection was observed in AAV-IL-30-treated mice. AAV-IL-27 treatment inhibited Th17 responses and induced multiple inhibitory pathways in T cells. Strikingly, we found that mice with established EAE was completely resistant to AAV-IL-27 or AAV-IL-30 treatment, and AAV-IL-27 treatment induced the expansion of CD11b + Gr-1 + myeloid cells that could produce multiple cytokines including Th17-promoting cytokines.
The complete prevention of EAE development in C57BL6 mice by AAV-IL-27 suggests potent protective mechanisms are activated. Indeed, we observed that AAV-delivered IL-27 inhibited the priming of Th17 cells, and induced T cell expression of Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 873 IL-10 and PD-L1. Inhibition of Th17 response is consistent with previous studies (37,38) using IL-27 as therapeutic, suggesting that AAV-IL-27-mediated inhibition of Th17 response contributes to the prevention of EAE. We also observed that AAV-IL-27 treatment inhibited the frequencies of GM-CSF-producing T cells in peripheral lymphoid organs in EAE protected mice, which is consistent with the known function of IL-27 in inhibiting GM-CSF production by T cells (17,24). Although high frequencies of IL-10-producing T cells were induced, our results suggest that AAV-IL-27-induced IL-10 production by T cells is not resp onsible for induction of T cell tolerance, since AAV-IL-27treatment induced complete protection of EAE development in  IL-10-deficient mice (Figure 4). IL-27-mediated PD-L1 expression in T cells has been shown to be sufficient for inducing T cell tolerance in a mouse model of human MS (10). In this study, we found that PD-L1 was induced in CD4 + T cells in the peripheral lymphoid organs (Figure 4) and in the CNS ( Figure 5D). However, despite the ability to enhance EAE development in AAV-ctrl treated mice, anti-PD-1 antibody treatment failed to break T cell tolerance induced by AAV-IL-27 ( Figure 4C). Thus, PD-L1 expression in T cells is not solely responsible for AAV-IL-27-mediated blockade of EAE development.
A striking finding in this study is that established EAE was resistant to AAV-IL-27 treatment, and in the CNS of AAV-IL-27-treated mice, CD4 + T cell production of key inflammatory cytokines such as IL-17 and GM-CSF were not affected or even  (Figures 5E,F). Lack of suppression of ongoing EAE by AAV-IL-27 could be due to the blood-brain barrier (BBB) prevented IL-27 access to the CNS or due to unresponsiveness of CNS-infiltrating T cells. However, these possibilities are highly unlikely. It is known that BBB is wide open during the CNS inflammation (48,49), and T cells in the CNS of mice with EAE expressed high levels of IL-27 receptor (Figure 5C). It is also unlikely that lack of suppression of ongoing inflammation is due to delayed production of IL-27 by AAV virus, since we observed that AAVmediated IL-27 production was efficient (by day 3 > 10 ng/mL of IL-27 can be detected in blood). More importantly, we found clear evidence that the CNS T cells from AAV-IL-27-treated mice were stimulated by IL-27, which is reflected by induction of PD-L1 expression, increased Th1 and decreased Treg responses ( Figure 5). Our results presented in Figures 6 and 7 suggest that the resistance of ongoing Th17-mediated CNS inflammation to IL-27 therapy could be due to the expansion of CD11b + Gr-1 + cells. It is well established that CD11b + Gr-1 + cells expand during EAE development (35,36). However, the role of this population of cells in EAE development is not clearly understood. Adoptive transfer experiment showed that these cells inhibited EAE development (35). However, other study clearly showed that these cells promoted Th17 responses via production of IL-1β, and depletion of this population of cells ameliorated EAE development (36).
In this study, we found that mice with established EAE had significant expansion of CD11b + Gr-1 + cells, and AAV-IL-27 treatment further expanded these cells. Moreover, we found that AAV-IL-27 treatment could directly induce the expansion of CD11b + Gr-1 + cells (Figure 6D), and adoptive transfer of CD11b + Gr-1 + cells from AAV-IL-27-treated mice enhanced EAE development ( Figure 7C). Thus, AAV-IL-27 therapy-induced expansion of CD11b + Gr-1 + cells enhances EAE development. Expansion of CD11b + Gr-1 + myeloid cells mainly occurred in the spleens and CNS but not in DLNs, suggesting that these Regulation of Autoimmune Encephalomyelitis by AAV-Delivered IL-27 Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 873 cells do not regulate T cell priming but mainly act at the effector phase of EAE development. This observation partially explains why EAE development was completely prevented despite some CD11b + Gr-1 + myeloid cell expansion was observed ( Figure 6C). The cytokine profiling of IL-27-expanded CD11b + Gr-1 + myeloid cells provides an explanation for why these cells enhance EAE development or confer resistance to IL-27 therapy. AAV-IL-27induced myeloid cells express multiple Th17-promoting cytokines including IL-1β, IL-6, IL-17 and IL-23. IL-1β, IL-6, and IL-23 have been well established as key cytokines for Th17 cell induction/ amplification and EAE development (23,36,50,51). Although we observed reduced IL-1β expression in FACS-sorted, IL-27expanded CD11b + Gr-1 + myeloid cells ( Figure 7A; reduced but still readily detectable by qPCR at 22 cycles), the overall expression of IL-1β increased in the CNS of AAV-IL-27-treated mice (Figure 5G), suggesting that more CD11b + Gr-1 + myeloid cells accumulated in the CNS and served as a major source of IL-1β. AAV-IL-27-induced CD11b + Gr-1 + myeloid cells also express S100A8/S100A9, which have been implicated in the inflamed CNS of mice with EAE (52) and shown to promote IL-1β and IL-6 production by immune cells (53). Thus, AAV-IL-27 therapy induces key cytokines for Th17 response in these cells, which could amplify the pre-existing Th17 cells during the effector phase of EAE. In addition to pro-inflammatory cytokines, we also observed that IL-27-expanded CD11b + Gr-1 + myeloid cells expressed high levels of IL-10, which could explain why high levels of pro-inflammatory cytokines detected in the CNS (Figure 5G) did not cause much worse disease ( Figure 5A). Lack of suppression of ongoing autoimmunity by AAV-IL-27 is consistent with the report (54) showing that IL-27 could not inhibit established Th17 responses, but appears to be inconsistent with other reports (9,13) demonstrating that systemic IL-27 inhibits T cell adoptive transfer EAE. At this stage, we do not know the reason for this inconsistency. AAV-mediated delivery of IL-27 is highly efficient and results in stable and high concentrations in the blood of the treated mice, and this is not easily achievable by systemic injection of IL-27 protein. It is thus necessary to determine if low and high concentrations of IL-27 induce T cell tolerance via different mechanisms. On the other hand, since adoptive transfer EAE involves a latent phase before EAE signs appear (55,56), suggesting that Th17 priming/differentiation in vivo is still needed for causing disease after T cell transfer. Thus, the initial stage of T cell adoptive transfer should not be considered as mice having an ongoing disease, and thus it is not surprising to see that IL-27 could suppress adoptive transfer EAE.
Taken together, our study suggests that systemic delivery of IL-27 can efficiently prevent EAE development and the priming of Th17 cells. However, the therapeutic potential of IL-27 may be limited by its failure to shut down established Th17 responses and reverse ongoing inflammation, presumably due to the expansion of CD11b + Gr-1 + myeloid cells. Moreover, the depletion of Treg cells adds additional risk for IL-27-based therapy of autoimmune diseases like MS.