Cbl-b restrains priming of pathogenic Th17 cells via the inhibition of IL-6 production by macrophages.

E3 ubiquitin ligase Cbl-b is involved in the maintenance of a balance between immunity and tolerance. Mice lacking Cbl-b are highly susceptible to experimental autoimmune encephalomyelitis (EAE), a Th17-mediated autoimmune disease. However, how Cbl-b regulates Th17 cell responses remains unclear. In this study, utilizing adoptive transfer and cell type-specific Cblb knockout strains, we show that Cbl-b expression in macrophages, but not T cells or dendritic cells (DCs), restrains the generation of pathogenic Th17 cells and the development of EAE. Cbl-b inhibits IL-6 production by macrophages that is induced by signaling from CARD9-dependent C-type lectin receptor (CLR) pathways, which directs T cells to generate pathogenic Th17 cells. Therefore, our data unveil a previously unappreciated function for Cbl-b in the regulation of pathogenic Th17 responses.

Introduction

Casitas-B-lineage lymphoma protein-b (Cbl-b), a Cbl family E3 ubiquitin ligase, encompasses multiple functional domains, including a protein tyrosine kinase-binding (TKB) domain, linker region, a RING (RING) finger (RF) domain, a proline-rich region, and a ubiquitin-associated region (Liu et al., 2014; Tang et al., 2019). The RF domain recruits E2 ubiquitin-conjugating enzymes which add ubiquitin to target substrates. The TKB domain recognizes specific phosphotyrosine residues on substrates for ubiquitin conjugation (Liu et al., 2014; Tang et al., 2019; Zhang, 2004). Previously, we and others have shown that Cbl-b is involved as a gatekeeper in the maintenance of a balance between immunity and tolerance (Bachmaier et al., 2000; Chiang et al., 2000; Heissmeyer et al., 2004; Jeon et al., 2004; Li et al., 2004; Zhang et al., 2002).

Genome-wide association studies (GWAS) have linked CBLB gene polymorphisms with patients with multiple sclerosis (MS) (Corrado et al., 2011; Sanna et al., 2010). CBLB polymorphisms can be associated with abnormalities in T cell function and responses to interferon-β therapy (Sturner et al., 2014). It was proposed that CBLB polymorphisms may affect the expression of CBL-B in T cells, thus mediating the disease process (Sturner et al., 2014). In support of these findings, loss of Cbl-b in mice facilitates the development of experimental autoimmune encephalomyelitis (EAE) (Chiang et al., 2000; Gruber et al., 2009), a mouse model of MS, and collagen-induced arthritis (Jeon et al., 2004), a mouse model of rheumatoid arthritis (RA), which are believed to be mediated at least in part by a pathogenic Th17 response. (Li et al., 2019; McGeachy et al., 2019; Sarkar et al., 2010) However, we have previously demonstrated that Cbl-b does not regulate Th1 and Th17 cell differentiation, but inhibits pro-allergic Th2/Th9 responses and allergic airway inflammation by targeting Stat-6 for ubiquitination and proteasome-mediated degradation (Qiao et al., 2014). Therefore, how Cbl-b regulates Th17 cell differentiation and Th17-mediated autoimmunity is currently unknown.

In this study, we utilized adoptive transfer and cell type-specific Cblb knockout strains to define the role of Cbl-b in priming of pathogenic Th17 cells and Th17-mediated autoimmunity using EAE as a model. We found that mice deficient for Cbl-b in macrophages, but not T cells and dendritic cells (DCs), develop severe EAE and a heightened pathogenic Th17 response, and that this is possibly achieved by aberrant IL-6 production by macrophages via CARD9-dependent CLR pathways, which facilitates the generation of pathogenic IL-17-producing CD4+ T cells.

Results

Mice lacking Cbl-b or expressing an inactive form of Cbl-b develop severe EAE and display heightened Th17 responses

Several previous studies showed that Cblb mice develop severe EAE (Chiang et al., 2000; Gruber et al., 2009) which is accompanied by a heightened Th17 response (Gruber et al., 2009). However, our previous study indicates that Cbl-b does not regulate Th17 but inhibits Th2/Th9 cell differentiation and Th2/Th9-mediated allergic airway inflammation (Qiao et al., 2014). To confirm whether Cbl-b inhibits EAE development and pathogenic Th17 responses via its E3 ubiquitin ligase activity, we immunized WT mice and mice lacking Cbl-b (Bachmaier et al., 2000) or expressing an E3 ubiquitin ligase dead mutation (C373A) (Oksvold et al., 2008) with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55) in Complete Freund’s Adjuvant (CFA) and monitored disease development for 23 days. In support of previous reports (Chiang et al., 2000; Gruber et al., 2009), mice with loss or inactivation of Cbl-b succumbed to severe disease (Figure 1A). This exacerbated disease was associated with hyper-proliferation of MOG35-55-specific T cells, and heightened production of IL-17, TNF-α, and IL-6 in the supernatants of draining lymph node (dLN) cell cultures (Figures 1B and 1C). We did not see any significant changes in the production of IL-1β, IL12p40, and TGF-β, but IL-10 was significantly reduced in the culture of dLN cells lacking Cbl-b or expressing the Cbl-b C373A mutant (Figure 1C). To determine whether Cbl-b deficiency or inactivation potentiates an antigen-specific Th17 response, we immunized WT, Cblb, and CblbC373A mice with MOG35-55 in CFA, and Th1 and Th17 responses were monitored in the dLNs on day 8 after immunization. Loss or inactivation of Cbl-b in mice elicited a heightened Th17 response, while the Th1 response was comparable among WT, Cblb, and CblbC373A mice (Figure 1D). Our data strongly indicate that Cbl-b inhibits Th17 responses and Th17-mediated autoimmunity in an E3 ubiquitin ligase-dependent manner.

Figure 1

Loss or inactivation of Cbl-b in mice leads to the development of severe clinical symptoms of EAE

(A) Clinical scores of WT, Cblb−/−, and CblbC373A mice (n = 5/group) immunized with MOG35-55 in CFA. ∗p < 0.05; Mann-Whitney U test.

(B and C) Ex vivo antigen-induced T cell proliferation and cytokine production of WT, Cblb−/−, and CblbC373A mice (n = 3/group for B, n = 5/group for C) immunized with MOG35-55 in CFA as described in A, determined by CFSE labeling and ELISA. ∗∗p < 0.01, ∗∗∗p < 0.001; Student t test.

(D) Flow cytometric analysis of Th1/Th17 responses in dLNs of WT, Cblb−/−, and CblbC373A mice (n = 3/group) on day 8 after immunization with MOG35-55 in CFA. ∗∗p < 0.01; Student t test. Bracket: the total number of gated CD4+ T cells. Data are one representative of three independent experiments.

Cbl-b expression in macrophages but not T cells or DCs is responsible for exacerbated EAE and heightened pathogenic Th17 responses

To determine whether T cell intrinsic Cbl-b is required for the inhibition of EAE and Th17 responses in vivo, we first adoptively transferred CD4+ T cells from WT and Cblb mice into Rag1 recipients, which do not have T and B cells, and then immunized with MOG35-55 in CFA. Surprisingly, Rag1 mice receiving CD4+ T cells from Cblb mice developed EAE similar to those receiving WT CD4+ T cells (Figure 2A). Further analysis showed that Th1 and Th17 cells in Rag1 mice receiving CD4+ T cells from Cblb mice were comparable to those receiving WT CD4+ T cells (Figure 2B). To further confirm this, we generated mice with a Cblb allele mutated by insertion of two loxP sites flanking parts of the exon 5 (Tang et al., 2020). We crossed Cblbf/f mice with Cd4 Cre mice to generate Cd4 Cre-Cblbf/f mice to specifically delete Cbl-b in the T cell lineage (Figure S1A). We immunized Cd4 Cre and Cd4 Cre-Cblbf/f mice with MOG35-55 in CFA and monitored disease development. Consistent with the data in Figure 2A, the EAE scores in Cd4 Cre and Cd4 Cre-Cblbf/f mice were comparable (Figure 2C), which was associated with similar levels of Th1 and Th17 responses (Figure 2D). In support of this data, ex vivo cytokine production including IL-6, IL-17A, IFN-γ, TNF-α, IL-12p40, TGF-β, IL-1β, IL-23, and IL-21 in the supernatants of dLN cell cultures stimulated with MOG35-55 was comparable in Cd4 Cre and Cd4 Cre-Cblb mice (Figure S2A). Therefore, our data collectively indicate that Cbl-b deficiency in T cells is not responsible for the exacerbated EAE and heightened Th17 response.

Figure 2

Cbl-b deficiency in myeloid cells is responsible for heightened EAE and Th17 responses

(A) Clinical scores of Rag1−/− mice receiving CD4+ T cells from WT (n = 8) and Cblb−/− (n = 7) mice, immunized with MOG35-55 in CFA.

(B) Flow cytometric analysis of Th1/Th17 responses in dLNs of Rag1−/−mice receiving CD4+ T cells from WT and Cblb−/− mice (n = 3/group) on day 8 after immunization with MOG35-55 in CFA.

(C) Clinical scores of Cd4 Cre (n = 10) and Cd4 Cre-Cblbf/f (n = 9) mice, immunized with MOG35-55 in CFA.

(D) Flow cytometric analysis of Th1/Th17 responses in dLNs of Cd4 Cre and Cd4 Cre-Cblbf/f mice (n = 3/group) on day 8 after immunization with MOG35-55 in CFA.

(E) Clinical scores of Rag1−/− (n = 11) and CblbRag1 (n = 10) mice receiving CD4+ T cells from WT mice, immunized with MOG35-55 in CFA. ∗p < 0.05; Mann-Whitney U test.

(F) Flow cytometric analysis of Th1/Th17 responses in dLNs of Rag1−/− and CblbRag1 mice (n = 4/group) receiving CD4+ T cells from WT mice on day 8 after immunization with MOG35-55 in CFA. ∗p < 0.05; Student t test.

(G) Clinical scores of LysM Cre and LysM Cre-Cblbf/f mice (n = 8/group), immunized with MOG35-55 in CFA. ∗∗p < 0.01; Mann-Whitney U test.

(H) Flow cytometric analysis of Th1/Th17 responses in dLNs of LysM Cre and LysM Cre-Cblbf/f mice (n = 3/group) on day 8 after immunization with MOG35-55 in CFA. ∗∗p < 0.01; Student t test. (I) Clinical scores of Cd11c Cre and Cd11c Cre-Cblbf/f mice (n = 6/group), immunized with MOG35-55 in CFA.

(J) Flow cytometric analysis of Th1/Th17 responses in dLNs of Cd11c Cre and Cd11c Cre-Cblbf/f mice (n = 3/group) on day 8 after immunization with MOG35-55 in CFA. Bracket: the total number of gated CD4+ T cells. Data are representative of two independent experiments.

To test whether Cbl-b deficiency in innate immune cells may be responsible for the phenotype observed in Cblb−/− mice, we crossed Cblb mice onto a Rag1 background to generate CblbRag1 mice which carry Cbl-b deficiency in innate immune cells. We adoptively transferred WT CD4+ T cells into Rag1 and CblbRag1 mice and monitored the disease development for 18 days. As shown in Figure 2E, CblbRag1 mice receiving WT CD4+ T cells developed severe EAE with an early onset. The severe disease was associated with an enhanced Th17 response (Figure 2F). To confirm this, we crossed Cblbf/f mice to LysM Cre and Cd11c Cre mice, respectively, to generate LysM Cre-Cblbf/f and Cd11c Cre-Cblb mice which delete Cbl-b in myeloid cells, and DCs, respectively (Figures S1B and S1C). We immunized LysM Cre and LysM Cre-Cblb, or Cd11c Cre, and Cd11c Cre-Cblb mice with MOG35-55 in CFA. Intriguingly, mice deficient for Cbl-b in myeloid cells (including monocytes, macrophages, and granulocytes), but not DCs, developed severe EAE with a significant increase in the Th17 response (Figures 2G–2J). This correlated with an increase in ex vivo cytokine production (IL-6, IL-17A, and TNF-α) by dLN cells from LysM Cre-Cblb mice stimulated with MOG35-55 (Figure S2B). Note that TGF-β production was not increased in mice lacking Cbl-b in T cells (Figure S2A), suggesting that the increased Th17 response is not due to altered TGF-β signaling. Interestingly, IL-10 production was lower in dLN cell culture in the presence of MOG35-55 of LysM Cre-Cblbf/f than LysM Cre mice (Figure S2B). In addition, mice deficient for Cbl-b in myeloid cells had similar Treg cells compared with control mice (Figures S3A and S3B). These results indicate that the severe EAE observed in mice deficient for Cbl-b in myeloid cells is not due to defective Treg cells. Furthermore, there were no differences in the percentage and numbers of Tregs in mice sufficient or deficient for Cbl-b in T cells (Figure S2B).

IL-6 produced by Cblb macrophages potentiates a pathogenic Th17 cell response

To define the cellular basis of this finding, we performed a co-culture experiment. We incubated CD4+ T cells from 2D2 mice, which have transgenic expression of a MOG35-55-specific TCR (Bettelli et al., 2003), together with bone-marrow-derived macrophages (BMDMs) or bone-marrow-derived dendritic cells (BMDCs) with MOG35-55 for 72 h in the presence of heat-killed M.tb. The cells were surface-stained with anti-CD4, and intracellularly stained with anti-IFN-γ and anti-IL-17. As shown in Figure 3A, 2D2 CD4+ T cells co-cultured with Cblb BMDMs, but not BMDCs, in the presence of heat-killed M.tb. skewed toward a Th17 phenotype compared to those co-cultured with WT BMDMs or BMDCs.

Figure 3

Hyper-production of IL-6 by Cblb−/− macrophages promotes a pathogenic Th17 response

(A) Th1/Th17 cell differentiation in co-cultures of 2D2 T cells with BMDMs or BMDCs from WT and Cblb−/− mice (n = 3/group) in the presence of heat-killed M.tb and MOG35-55. ∗∗∗p < 0.001; Student t test.

(B) Th1/Th17 cell differentiation in co-cultures of 2D2 T cells with BMDMs from WT and Cblb−/− mice (n = 3/group) in the presence of heat-killed M.tb and MOG35-55 with or without pretreatment with mitomycin C. ∗∗∗p < 0.001; Student t test.

(C) Flow cytometric analysis of IL-6-producing cells of dLNs of WT and Cblb−/− mice (n = 3/group) on day 8 after immunization with MOG35-55 in CFA, and stimulated with PMA/ionomycin. The gating strategy and representative plots see Figure S5. ∗∗p < 0.01; Student t test.

(D) ELISA of pro-inflammatory cytokines (TNF-α, IL-6, IL-10, IL-12p40, IL-1β, TGF-β, IL-23, and IL-21) by BMDMs from WT and Cblb−/− mice (n = 5/group) stimulated with heat-killed M.tb. ∗p < 0.05, ∗∗∗p < 0.001; Student t test.

(E) Th1/Th17 cell differentiation in co-cultures of 2D2 CD4+ T cells with BMDMs from Cblb−/− mice that had been treated with Il6 or control siRNA. IL-6-producing macrophages of Cblb−/− mice treated with Il6 (n = 4) or control siRNA (n = 4) were also determined. The mean fluorescent intensity (MFI) determined by flow cytometry indicates the expression level of IL-6 in CD4− macrophages of Cblb−/− mice treated with Il6 or control siRNA. ∗∗∗p < 0.001; Student t test.

(F) Clinical scores of Cblb−/− mice immunized with MOG35-55 in CFA in the treatment with or without Il6 siRNA (n = 5) or control siRNA (n = 5) on day 1, 3, and 7 via tail vein injection. ∗∗p < 0.01; Mann-Whitney U test.

(G) Flow cytometric analysis of Th1/Th17 responses and IL-6-producing cells in dLN cells from Cblb−/− mice treated with Il6 (n = 3) or control siRNA (n = 3) via tail vein injection as in F on day 8 after immunization with MOG35-55 in CFA. MFI determined by flow cytometry indicates the expression level of IL-6 in F4/80+ cells from dLNs of Cblb−/− mice treated with Il6 (n = 3) or control siRNA (n = 3). ∗p < 0.05; Student t test.

(H) Clinical scores of Cblb mice immunized with MOG35-55 in CFA in the treatment with or without Il6 siRNA (n = 5) or control siRNA (n = 5) on day 8, 11, and 15 via tail vein injection. Bracket: the total number of gated CD4+ T cells. Data are representative of three independent experiments (A, B, E, F, and G) and representative of two independent experiments (C-D).

Macrophages can function both as antigen-presenting cells (APCs) and secrete pro-inflammatory cytokines such as IL-6, which can regulate a pathogenic Th17 cell response (Heink et al., 2017). To dissect these two functions of macrophages in Th17 cell differentiation, we pre-treated WT and Cblb BMDMs with mitomycin C which inactivates macrophages but maintains their antigen presentation function. Pre-treating Cblb−/− macrophages with mitomycin C completely abrogated both CD4+IFN-γ+ and CD4+IL-17+ T cells in the presence of heat-killed M.tb. (Figure 3B), suggesting that the pro-inflammatory cytokines produced by Cblb−/− macrophages potentiate the Th17 cell response. Taken together, our data collectively indicate that the cytokines produced by Cblb−/− BMDMs strongly induced a pathogenic Th17 response.

IL-6 is a key cytokine to initiate Th17 cell differentiation (Heink et al., 2017; Korn et al., 2008). Several cellular sources including T cells, macrophages, and DCs can produce IL-6 (Couper et al., 2008; Heink et al., 2017). To further confirm whether Cbl-b deficiency in macrophages leads to heightened IL-6 production, we immunized WT and Cblb−/− mice with MOG35-55 in CFA. Eight days later we analyzed IL-6-producing macrophages (CD11b+F4/80+), DCs (CD11b−CD11c+ and CD11b+CD11c+), monocytes/neutrophils (CD11b+Ly6C+Ly6G−/CD11b+Ly6C−Ly6G+), CD3−CD19+ B cells, and CD3+CD4+ T cells in the dLNs. Interestingly, we observed that there were significantly more IL-6-producing macrophages from Cblb mice than WT mice (Figure 3C). In contrast, there was no significant difference observed in IL-6-producing T cells, B cells, DCs, monocytes, or neutrophils from WT and Cblb mice (Figure 3C), further confirming that IL-6 derived from macrophages lacking Cbl-b is the major source that mediates a pathogenic Th17 response during EAE induction. Consistent with this finding, BMDMs derived from Cblb mice produced significantly more TNF-α and IL-6, and lesser amounts of IL-10, upon stimulation with heat-killed M.tb. in vitro (Figure 3D). The decreased IL-10 in Cblb−/− macrophages may be due to enhanced IL-6 production. Furthermore, we previously showed that there is no significant difference in TNF-α and IL-6 production by WT and Cblb BMDMs upon LPS stimulation (Xiao et al., 2016). These findings are also consistent with our data presented Figure S2B in which IL-6 and TNF-α production was significantly increased in the supernatants collected from dLN cell culture from LysM Cre-Cblb but not Cd4 Cre-Cblb mice in an ex vivo MOG35-55 recall response.

To prove whether heightened IL-6 by Cblb−/− BMDMs facilitates a pathogenic Th17 response, we knocked down the Il6 gene in Cblb−/− BMDMs by Il6 siRNA, and then co-cultured 2D2 T cells with Cblb−/− BMDMs in the presence of MOG35-55 and heat-killed M.tb for 72 h. Silencing the Il6 gene in Cblb−/− BMDMs markedly inhibited the Th17 cell response (Figure 3E). In further support of this data, treating Cblb−/− mice with Il6 siRNA significantly ameliorated EAE disease activity (Figure 3F) and inhibited the Th17 response (Figure 3G). In addition, we treated siRNA during the onset stage (8 days after antigen immunization) and investigated the effect of Il6 siRNA against EAE. In contrast to the data presented in Figure 3F, delayed treatment of Cblb−/− mice with Il6 siRNA failed to suppress EAE progression (Figure 3H). Our data are consistent with a previous report that IL-6 blockade by an anti-IL-6 monoclonal antibody inhibits the induction of antigen-specific Th1 and Th17 cells in EAE (Serada et al., 2008).

CNS macrophages, but not neutrophils and microglia, from Cblb mice with EAE produce more IL-6

To determine whether IL-6-producing macrophages in the CNS of Cblb−/− mice contribute to disease progression, and whether neutrophils or microglia also play a role in disease progression, we immunized WT and Cblb−/− mice with MOG35-55 in CFA, and 16–18 days later mice were sacrificed, and leukocytes from the spinal cords and brains were assessed for IL-6-producing cells in the CNS. As shown in Figure 4, IL-6-producing macrophages, but not neutrophils and microglia, were significantly enhanced in Cblb−/− mice compared to WT controls. These data further support a pathogenic role of IL-6 by macrophages in disease development.

Figure 4

CNS macrophages, but not neutrophils and microglia, from Cblb mice with EAE produce more IL-6

Flow cytometric analysis of IL-6-producing macrophages (CD11b+Ly6C+Ly6G), neutrophils(CD11b+Ly6G+Ly6C+), and microglia (CD11b+Ly6G−Ly6C−TMEM119+) cells of CNS of WT and Cblb−/− mice (n = 4/group) on day 17 after immunization with MOG35-55 in CFA, and stimulated with PMA/ionomycin. ∗∗p < 0.01; Student t test. Data are representative of two independent experiments.

Deficiency for Dectin-2 but not Dectin-1 attenuates EAE disease activity in Cblb−/− mice

One major component in CFA is heat-killed Mycobacterium tuberculosis (M.tb.). M.tb. possess various cell wall components that influence host immune responses, such as trehalose-6,6’-dimycolate (TDM), mycolate, phosphatidyl-myo-inositol mannosides (PIMs), lipomannnan (LM), and lipoarabinomannan (LAM) (Wolfe et al., 2010). Dectin-2 is a direct receptor for mannose-capped LAM (Man-LAM) in M.tb. Mice lacking Dectin-2 (Clec4n) are resistant to EAE when heat-killed M.tb. is replaced with LAM (Yonekawa et al., 2014). Dectin-3 and Mincle recognize the Mycobacterial cord factor, TDM, and induce potent innate immune responses (Ishikawa et al., 2009; Matsunaga and Moody, 2009; Miyake et al., 2013; Zhao et al., 2014). Mice deficient for Dectin-3 (Clec4d) are resistant to EAE induction when using TDM as an adjuvant (Miyake et al., 2013). We previously showed that Dectin-2 and -3 are the targets for Cbl-b during fungal infections (Xiao et al., 2016). Since Dectins, in particular Dectin-2 and Dectin-3, have been shown to be required for the induction of EAE via their interactions with their ligands in M.tb., a major component in CFA, we reasoned that the increased susceptibility to EAE and pathogenic Th17 response in Cblb−/− mice is due to aberrant expression of these Dectins, which correlates with our previous observation that Cbl-b targets Dectin receptors for ubiquitination and lysosome-mediated degradation (Xiao et al., 2016). Indeed, the expression of Dectin-2, -3, and Mincle was heightened in mice lacking Cbl-b (Figure S4).

To test this, we first determined whether loss of Dectin-2 attenuates the development of EAE in mice lacking Cbl-b, by generating CblbClec4n mice. We also used Clec7a and CblbClec7a mice as controls, which are deficient for Dectin-1 or Dectin-1 and Cbl-b, respectively. In support of our prediction, loss of Dectin-2 attenuated the clinical scores in Cblb−/− mice to the levels of WT and Clec4n mice (Figure 5A), which correlated with a decreased Th17 response (Figure 5B). Surprisingly, Clec7a mice developed severe EAE and an enhanced Th17 response (Figures 5A and 5B), and Cbl-b deficiency did not worsen the disease in Clec7a mice (Figures 5A and 5B). Given that we have shown that Cbl-b targets Dectin-1 for ubiquitination and lysosome-mediated degradation (Xiao et al., 2016), these data suggest that Cbl-b and Dectin-1 inhibit the development of EAE via different mechanisms. Although both Dectin-3 and Mincle are receptors for TDM, constitutive expression of Dectin-3 in myeloid cells is required for Mincle expression in response to TDM adjuvant (Miyake et al., 2013). Furthermore, TDM-induced EAE is almost completely dependent on Dectin-3, which is associated with a defective Th17 response (Miyake et al., 2013). Therefore, it is possible that both Dectin-2 and Dectin-3/Mincle contribute to severe EAE in Cblb−/− mice. This notion is supported by the fact that congenic rats expressing lower levels of Dectin-3 and Mincle on myeloid cells exhibit a drastic reduction in EAE incidence and severity, which correlates with an impaired Th17 response in CNS (N'Diaye et al., 2020). Indeed, mice deficient for CARD9, the major adaptor protein which forms a complex with Bcl-10 and MALT-1 to induce the activation of NF-κB (Tang et al., 2018a), developed delayed and ameliorated EAE (Figure 5C), with a defective Th17 response (Figure 5D) and impaired macrophage-derived IL-6 (Figure 5E).

Figure 5

CARD9-mediated Dectin receptor signaling contributes the clinical symptoms of Cblb−/− mice with EAE and pathogenic Th17 response

(A) Clinical scores of WT, Cblb, Clec4n, Clec7a, CblbClec4n, and CblbClec7a mice (n = 5/group) immunized with immunized with MOG35-55 in CFA. ∗∗∗p < 0.001, Mann-Whitney U test.

(B) Flow cytometric analysis of Th1/Th17 responses in dLNs of WT, Cblb, Clec4n, Clec7a, CblbClec7a, and CblbClec4n mice (n = 4/group) on day 8 after immunization with MOG35-55 in CFA. ∗∗p < 0.01, Student t test.

(C) Clinical scores of WT and Card9 mice (n = 7/group) immunized with MOG35-55 in CFA. ∗∗p < 0.01, Mann-Whitney U test.

(D) Flow cytometric analysis of Th1/Th17 responses in dLNs WT and Card9 mice (n = 5/group) on day 8 after immunization with MOG35-55 in CFA. ∗∗p < 0.01, Student’s t test.

(E) Flow cytometric analysis of Th1/Th17 responses in dLNs WT and Card9 mice (n = 3/group) on day 8 after immunization with MOG35-55 in CFA. ∗p < 0.05, Student’s t test. Data are three independent experiments for A and B, and two independent experiments for C, D, and E.

Discussion

It has been well documented that mice deficient for Cbl-b develop severe EAE (Chiang et al., 2000; Gruber et al., 2009), and that CBLB polymorphisms are associated with MS disease activity (Corrado et al., 2011; Sanna et al., 2010; Sturner et al., 2014). However, the cellular and molecular mechanisms are poorly defined. In this study, we found that Cbl-b expression in myeloid cells, but not T cells, is responsible for restraining the priming of a pathogenic Th17 response, and this process is achieved by controlling the production of IL-6 by macrophages mediated by the signaling pathways of CARD9-dependent CLRs.

CBLB polymorphisms have been shown to associate with several autoimmune diseases in humans including MS, type 1 diabetes, and lupus (Bergholdt et al., 2005; Corrado et al., 2011; Doniz-Padilla et al., 2011; Sanna et al., 2010; Sturner et al., 2014). It was believed that Cbl-b deficiency leads to the impairment of T cell tolerance, thus resulting in aberrant autoreactive T cell proliferation (Bachmaier et al., 2000; Chiang et al., 2000; Jeon et al., 2004; Liu et al., 2014; Tang et al., 2018b). To our surprise, our previous data strongly indicate that Cbl-b does not inhibit Th17 cell differentiation, rather suppresses pro-allergic Th2/Th9 responses and allergic airway inflammation (Qiao et al., 2014). Our findings suggested that Cbl-b deficiency in innate immune cells may be responsible for heightened Th17 responses and severe EAE observed by other groups. Indeed, using Rag1 or CblbRag1 mice in combination with adoptive transfer, and conditional knockout strains specifically lacking Cbl-b in T cells, myeloid cells, and DCs, we demonstrate that loss of Cbl-b in macrophages leads to the aberrant production of pro-inflammatory cytokines which promote the differentiation of T cells into the pathogenic Th17 lineage and the development of EAE (Figure 2). This notion is supported by our recent study that Cblb−/− macrophages produce significantly more IL-6 and TNF-α upon infection with Candida albicans yeast and hyphal forms (Xiao et al., 2016).

IL-6 is a pleiotropic cytokine with significant functions in the regulation of the immune system. As a potent pro-inflammatory cytokine, IL-6 appears to play key roles in genetically diverse autoimmune diseases such as relapsing remitting MS, RA, and systemic lupus erythematosus (Choy et al., 2020; Kang et al., 2019). GWAS have confirmed that CBLB gene polymorphisms are associated with MS (Corrado et al., 2011; Sanna et al., 2010). It is possible that CBLB polymorphisms lead to increased IL-6 production by macrophages in patients with MS, which in turn leads to increased pathogenic Th17 cells and ultimately aggravates the disease. Indeed, macrophages, but not T cells, B cells, monocytes, DCs, or neutrophils from Cblb−/− mice during EAE induction produce more IL-6 than macrophages from WT mice (Figure 3C). By co-culturing 2D2 T cells with WT and Cblb−/− BMDMs or BMDCs, in the presence of heat-killed M.tb. and MOG35-55 peptide, we found that Cblb−/− BMDMs, but not BMDCs, facilitate 2D2 T cells to differentiate into Th17 cells which are abrogated when they are treated with mitomycin C (Figure 3B). This biased Th17 cell differentiation is most likely due to increased production of IL-6 by macrophages because 1) more IL-6 production was observed in Cblb−/− BMDMs stimulated with heat-killed M.tb (Figure 3D); 2) silencing the Il6 gene in Cblb−/− BMDMs by Il6 siRNA abrogates the heightened Th17 cell differentiation in vitro (Figure 3E); and 3) in vivo delivery of Il6 siRNA inhibits the development of EAE in Cblb−/− mice (Figure 3F). Inhibition of IL-6 in Cblb−/− mice at the induction phase, but not after disease onset, suppresses disease progression (Figure S5), suggesting that targeting IL-6 to treat patients with active MS may not work. This notion is supported by the failure of anti-IL-6R treatment to suppress established EAE (Serada et al., 2008). A recent study reported that a Stat3 inhibitor ameliorates CNS autoimmunity in EAE by restoring the balance of Th17 vs. regulatory T cells (Tregs) (Aqel et al., 2021). IL-6, signaling through Stat3, induces the development of highly encephalitogenic Th17 cells (Das et al., 2009; Ghoreschi et al., 2010). IL-23, an inflammatory cytokine that is critical for EAE development and progression, signals through Stat3 and expands effector/memory myelin-specific Th17 cells in EAE mice (Ghoreschi et al., 2010; Langrish et al., 2005). Therefore, targeting signaling molecules downstream of IL-6/IL-23 such as Stat3 or IL-6 and IL-23 themselves may represent effective therapeutic strategies to treat MS via rebalancing pathogenic Th17:Tregs.

The development of MS involves both genetic and environmental factors such as viral infection. Several lines of evidence suggest that MS may be triggered by microbial infections. Indeed, fungi and bacteria have been directly identified in the CNS (Alonso et al., 2018). Indeed, CBLB variants have been shown to be associated with MS (Corrado et al., 2011; Sanna et al., 2010). M.tb. contain many TLR and CLR ligands (Faridgohar and Nikoueinejad, 2017; Miyake et al., 2013; Wassermann et al., 2015; Yonekawa et al., 2014) which can trigger inflammatory responses by innate immune cells. Since Dectin-2 deficiency fails to completely inhibit the development of EAE and Th17 responses in Cblb−/− mice (Figures 50A and 5B), it is possible that other CLRs such as Dectin-3 and Mincle may also be involved in the induction of the Th17 response. Indeed, mice deficient for Dectin-3 develop ameliorated EAE (Miyake et al., 2013), and congenic rats expressing lower levels of Dectin-3 and Mincle on myeloid cells exhibit a drastic reduction in EAE incidence and severity, which correlates with an impaired Th17 response in CNS (N'Diaye et al., 2020). Our data are further supported by the fact that Card9−/− mice develop significantly attenuated EAE and impaired pathogenic Th17 responses (Figures 5C and 5D). Our data are consistent with a recent report in which CARD9 has been shown to be required for the inflammatory pathology in experimental autoimmune uveoretinitis and Th17 responses (Lee et al., 2016). Note that our data indicate that Card9−/− mice still develop mild EAE (Figure 5C), suggesting that other signaling pathways may also be involved in EAE induction.

It was reported that Dectin-1 limits EAE and promotes myeloid cell-astrocyte crosstalk via a CARD9-independent expression of oncostatin M (Deerhake et al., 2021). However, the Th17 response was not altered in mice lacking Dectin-1 in this study (Deerhake et al., 2021) which is different from the data of our current study (Figures 5A and 5B). Nevertheless, our data, and those published by others (Deerhake et al., 2021), suggest that Dectin-1 may inhibit EAE development independently of CARD9. Therefore, the cellular and molecular mechanisms by which Dectin-1 inhibits EAE development are still not fully defined, and is currently under investigation in our laboratory.

In conclusion, we have provided compelling evidence that Cbl-b inhibits Th17 responses and Th17-mediated autoimmunity, and this inhibition is achieved by the suppression of IL-6 production by macrophages which directs T cells to differentiate into the Th17 cell lineage. Therefore, our study unveils a previously unsolved cellular mechanism how Cbl-b inhibits pathogenic Th17 responses.

Limitations of the study

Although GWAS studies have linked CBLB gene polymorphisms with patients with multiple sclerosis, it is still unknown whether CBLB gene polymorphisms lead to aberrant production of IL-6 by macrophages in patients with multiple sclerosis. In addition, how the interaction of Cbl-b-deficient macrophages with pathogenic Th17 cells plays a role in the pathogenesis of CNS demyelination needs to be explored in future studies.

STAR★Methods

Key resources table

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Jian Zhang (jian-zhang@uiowa.edu).

Materials availability

Requests for further information or materials should be directed to the lead contact.

Experimental model and subject details

Mice

C57BL/6, 2D2 TCR transgenic, Card9−/−, Clec7a, Cd4 Cre, LysM Cre, Cd11c Cre, and B6.129S7-Rag1tm1Mom/J (Rag1−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Cblb mice were kindly provided by Dr. Josef M. Penninger (University of Toronto; Toronto, ON, Canada). Cblb mice were described previously (Oksvold et al., 2008). Clec4n mice (Saijo et al., 2010) were obtained from Dr. Yoichiro Iwakura (Tokyo University of Science; Chiba, Japan). Cblb−/− mice were crossed with Rag1−/−, Clec4n, and Clec7a mice to generate CblbRag1, CblbClec4n, and CblbClec7a mice.

The mouse strain carrying the Cblb allele was generated at the Ohio State University Genetically Engineered Mouse Modeling Core Facility by standard embryonic stem (ES) cell technology as described previously (Tang et al., 2020). The ES clone EPD0703_2_B11 was acquired from the International Knockout Mouse Phenotyping Consortium (IMPC project #79117). Prior to microinjection, the identity of the targeted embryonic stem cells was verified by 5′ long-range PCR using a primer external to the targeting vector. Chimeric males were bred to C57BL/6 Albino females and germline transmission was verified by PCR to detect the mutant together with the wild-type allele in the F1 heterozygous mice. Prior to utilization of the strain for experiments, mice were crossed to a Flipe ubiquitous strain (ACTB:FLPe B6J, JAX strain # 005703) (Rodriguez et al., 2000) to eliminate the lacZ/neo cassette and obtain the clean tm1c allele according to the breeding schemes recommended by the IMPC. The LoxP flanked Cblb allele (Cblb) was crossed to Cd4 Cre, LysM Cre, and Cd11c Cre mice to specifically delete Cbl-b in T cells, myeloid cells including macrophages, and DCs. All mice were bred and housed under specific pathogen–free (SPF) conditions. The experimental protocols followed National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committees of The Ohio State University and the University of Iowa. All mice were used for experiments at ages of 8–12 weeks, and both male and female mice were used in this study.

Method details

EAE induction

Various groups of mice at 8-12 weeks were subcutaneously (s.c.) injected over four sites in the flank with 100 μL of emulsified IFA supplemented with 200 μg of MOG33–55 and 500 μg of heat-killed M.tb as previously described (Ouyang et al., 2019; Xiao et al., 2015). Three hundred ng pertussis toxin (List) per mouse in PBS was injected intraperitoneally (i.p.) at the time of immunization and 48 h later. The mice were monitored daily for clinical signs of EAE, and were scored on a scale of 0–5 (Miller & Karpus, 2007): 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hindlimb weakness; 3, severe hindlimb weakness; 4, complete hindlimb paralysis; and 5, quadriplegia or premoribund state.

CD4+ isolation and in vitro CD4+ T cells proliferation

CD4+ T cells from WT, Cblb and Cblb mice immunized with MOG35-55 in CFA were isolated by the CD4+ T cell Isolation Kit (Miltenyi Biotec). The CD4+ T cells were labeled with CFSE and cultured with 20 μg/mL MOG35–55 in the presence of irradiated splenocytes depleted of T cells for 72 h. The proliferation rates of CD4+ T cells were determined by CFSE dilution. The supernatants collected from these cultures were subjected for ELISA for IL-17A, IFN-γ, IL-12p40, IL-6, IL-10, IL-1β, TNF-α, TGF-β, IL-21 and IL-23 using sandwich ELISA kits (BioLegend). All the procedures were performed according to the manufacturer’s instructions.

Detection of Th1 and Th17 responses during EAE induction

For detection of Th1 and Th17 responses, dLN cells collected from various groups of mice immunized with MOG35-55 in CFA on day 8 were stimulated with 20 μg/mL MOG35-55 in culture medium for 3 days. The supernatants collected from these cultures were subjected for ELISA for IL-17A, IFN-γ, IL-12p40, IL-6, IL-10, IL-1β, TNF-α, TGF-β, IL-21 and IL-23 using the sandwich ELISA kits (BioLegend). The cells were restimulated with 50 ng/mL PMA and 750 ng/mL ionomycin for 4 h. The cells were surface-stained with anti-CD4, and intracellularly stained with anti-IFN-γ or anti-IL-17, respectively. The CD4+IFN-γ+ and CD4+IL-17+ cells were determined by flow cytometry as previously described (Qiao et al., 2012, 2014; Xiao et al., 2015; Ying et al., 2010).

CD4+ T cells isolation and adoptive transfer for EAE induction

CD4+ T cells from WT and Cblb mice were isolated by the CD4+ T cell Isolation Kit (Miltenyi Biotec) and injected into Rag1−/− mice by i.v. at 5 × 106 cells/mouse. In a separate experimental setting, CD4+ T cells from WT mice were adoptively transferred into Rag1−/− and Rag1−/−Cblb−/− mice, respectively. Thirty days later, the recipient mice were immunized with MOG35-55 in CFA.

Generation of bone marrow-derived macrophages (BMDMs) and bone marrow-derived DCs (BMDCs)

Erythrocyte-depleted mouse bone marrow (BM) cells were cultured from WT and Cblb mice strains and were harvested from the femurs and tibias of mice. Cells were cultured in DMEM containing 10% FBS and 30% conditioned medium from L929 cells expressing M-CSF as described (Xiao et al., 2016). After one week of culture, non-adherent cells were removed, and adherent cells were 80-90% F4/80+CD11b+ as determined by flow cytometric analysis.

For BMDC generation, bone marrow cells were cultured in RPMI supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/mL streptomycin, 0.05 mM β-mercaptoethanol and 20 ng/mL GM-CSF (R&D Systems, Minneapolis, MN). The cultures were usually fed every 2 days by gently swirling the plates, aspirating 75% of the medium, and adding back fresh medium with GM-CSF. After 7-8 days, DMDCs were enriched by a DC enrichment kit (Miltenyi Biotec).

Co-culture of 2D2 T cells with BMDMs or BMDCs

CD4+ T cells from 2D2 mice were incubated with BMDMs or BMDCs pre-pulsed with 20 μg/mL of MOG35-55 peptide overnight in the presence of 20 μg/mL of heat-killed M.tb at a 1:1 ratio. On day 3, the cells were stimulated with PMA/ionomycin for 4 h, surface-stained with anti-CD4, and intracellularly stained with anti-IFN-γ and anti-IL-17 and analyzed by flow cytometry.

Treatment of BMDMs with mitomycin C

For mitomycin C treatment, BMDMs from WT and Cblb−/− mice were suspended at a concentration of 2 × 107 cells/ml in PBS, and treated with or without Mitomycin C (50 μg/mL) at 37°C for 20 min. WT and Cblb−/− BMDMs with or without Mitomycin C treatment were co-cultured with 2D2 CD4+ T cells in the presence of MOG35-55 as described above.

Determination of IL-6-producing cells by flow cytometry

WT and Cblb−/− mice were immunized with MOG35-55 in CFA. dLN cells were collected on day 8, and restimulated with PMA/ionomycin. Cells were then collected, washed, and resuspended in staining buffer (1% BSA in PBS). The cells were incubated with mAbs to various cell-surface markers for 30 min at 4°C. After washing twice with staining buffer, cells were fixed and permeabilized using Cytofix/Cytoperm solution (BD Bioscience) for 20 min at 4°C. Cells were stained for intracellular IL-6 for 30 min at 4°C. IL-6-producting cells were determined by flow cytometry. Flow cytometric analysis was performed to evaluate IL-6-producing cells in macrophages (CD11b+F4/80+), DCs (CD11b−CD11c+ and CD11b+CD11c+), monocytes/neutrophils (CD11b+Ly6C+Ly6G−/CD11b+Ly6C−Ly6G+), CD3−CD19+ B cells, and CD3+CD4+ T cells.

In vitro Knockdown experiments

Il6 Accell siRNA or non-sense siRNA were obtained from Dharmacon. BMDMs were plated in 12 wells and transiently transfected with 2 μg of siRNAs using Lonza Nucleofector reagent according to the Manufacturer’s instruction. 36 h later, cells were harvested. IL-6 expression was determined by flow cytometry.

In vivo delivery of Il6 siRNA

In vivo delivery of siRNA was described previously (Tang et al., 2020; Xiao et al., 2016) with some modifications. In brief, Cblb mice were treated with Il6 Accell siRNA (L-043739-00-0005, Dharmanon) at 10 nM/kg/mouse) or a non-sense siRNA by tail vein injection one day before immunization (Day −1) with MOG35-55 in CFA, and then twice (Day 3 and Day 7) after immunization. The Il6 Accell siRNA and non-sense siRNA were dissolved by sterile PBS, and the final dose of injection is 100 μL per mouse. In a parallel experiment, the dLNs from Cblb−/− mice received Il6 Accell siRNA or the control siRNA were collected at day 10. The cells were surface-stained with anti-CD4,anti-CD11B, anti-F4/80,and intracellularly stained with anti- IFN-γ or anti-IL-17、anti- IL-6, anti- IFN-γ or anti-IL-17. The expression of CD4+IFN-γ+, CD4+IL-17+, and CD11b+F4/80+IL-6+ populations was determined by flow cytometry.

Isolation of CNS infiltrating cells

Spinal cord was removed by insufflation using 20 mL 4°C RPMI expressed through a 19G needle. The spinal cord was transferred to 10 mL fresh, cold RPMI and homogenized by using the 5 mL syringe. Cells were pelleted by centrifugation at 300×g for 5 min at 4°C and the supernatant was discarded. The cells were resuspended in 1 mL digestion solution (PBS with Calcium and Magnesium, 1.5 mg/mL Collagenase II, 50 μg/mL DNase I) by using vortexer and incubated for 30 min, 37°C in water bath. 30–70% colloid Percoll was used to isolate CNS infiltrating cells. The cells were carefully harvested from the interphase 30–70% Percoll fraction and were transferred into new 15 mL Falcon tube containing 8mL PBS/FCS. Cells were pelleted by centrifugation at 900×g, 5 min, 4°C and the supernatant was discarded. The isolated cells were further used for detection of IL-6-producing macrophages, neutrophils, and microglia.

Quantification and statistical analysis

Data analysis and statistical analysis

GraphPad Prism Software (San Diego, CA, USA) was utilized for statistical analysis. A two-tailed Student’s t-test was applied for statistical comparison of two groups or, where appropriate and a Mann-Whitney U test for nonparametric data (EAE scoring). Differences were considered significant at p < 0.05. No animals were excluded from the analysis. Mice were allocated to experimental groups based on their genotypes and were randomized within their sex- and age-matched groups. No statistical method was used to predetermine sample size. It was assumed that normal variance occurs between the experimental groups.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
anti-mouse CD28 (37.51)	BD Biosciences	Cat#: 553294; RRID:AB_394763
anti-CD3 (145-2C11)	BD Biosciences	Cat#: 550275; RRID:AB_393572
Mouse GM-CSF	R&D Systems	Cat#: 415-ML-010/CF
hamster IgG isotypic controls	BD Biosciences	Cat#: 553951; RRID:AB_395155
anti-mouse CD4-PerCP	BioLegend	Cat#: 100432; RRID:AB_893323
anti-mouse CD4-FITC	BioLegend	Cat#: 100406; RRID:AB_312691
anti-mouse CD4-allophycocyanin/Cy7	BioLegend	Cat#: 100414; RRID:AB_312699
anti-mouse CD3-allophycocyanin	BioLegend	Cat#: 100236; RRID:AB_2561456
anti-mouse CD11b-allophycocyanin	BioLegend	Cat#: 101212; RRID:AB_312795
anti-mouse CD11c-FITC	BioLegend	Cat#: 117306; RRID:AB_313775
anti-mouse F4/80-PE/Cy7	BioLegend	Cat#: 123114; RRID:AB_893478
anti-mouse Ly6G-Brilliant Violet 421	BioLegend	Cat#: 127628; RRID:AB_2562567
anti-mouse Foxp3-PE	BioLegend	Cat#: 320008; RRID:AB_492980
anti-mouse CD25-allophycocyanin	BioLegend	Cat#: 101910; RRID:AB_2280288
anti-mouse IL-17A-PE	BioLegend	Cat#: 506904; RRID:AB_315464
anti-mouse IFN-γ-allophycocyanin	BioLegend	Cat#: 505810; RRID:AB_315404
anti-mouse IL-6-FITC	eBioscience	Cat#: 11-7061-41; RRID:AB_1633408
anti-mouse CD19-Brilliant Violet 786	BD Biosciences	Cat#: 563333; RRID:AB_2738141
anti-mouse Ly6C-PerCP/Cy5.5	BD Biosciences	Cat#: 560525; RRID:AB_1727558
anti-mouse CD3-PE/Cy5	BioLegend	Cat#: 100273; RRID:AB_2894410
anti-mouse F4/80-PerCP/Cy5.5	BioLegend	Cat#: 157318; RRID:AB_2894410
anti-mouse CD11b-Pacific Blue	BioLegend	Cat#: 101224; RRID:AB_755986
anti-mouse TMEM119-PE/Cy7	ThermoFisher	Cat#: 25-6119-82; RRID:AB_2848312
anti-mouse IL-6-APC	BioLegend	Cat#: 504508; RRID:AB_10694868
anti-mouse CD45.2-Brilliant Violet 711	BioLegend	Cat#: 109847; RRID:AB_2616859
anti-mouse CD11b-Brilliant Violet 605	BioLegend	Cat#: 101257; RRID:AB_2565431
anti-mouse Ly-6C-PE	BioLegend	Cat#: 128008; RRID:AB_1186132
anti-mouse Ly-6G-FITC	BioLegend	Cat#: 127606; RRID:AB_1236494
anti-mouse Dectin-2-FITC	Miltenyi Biotec	Cat#: 130-125-214; RRID:AB_2889657
anti-mouse Dectin-1-PE	BioLegend	Cat#: 144304; RRID:AB_2561501
anti-mouse Dectin-3-APC	BioLegend	Cat#: 360206; RRID:AB_2563154
anti-mouse B220-PE/Cy7	BioLegend	Cat#: 103222; RRID:AB_313005
anti-mouse Cbl-b (H-121)	Santa Cruz	Cat#: sc-1704; RRID:AB_2070711
Chemicals, peptides, and recombinant proteins
Mycobacterium tuberculosis strain H37Ra	Difco	Cat#: 231141
carboxyfluorescein succinimidyl ester (CFSE)	BioLegend	Cat#: 422701
Pertussis toxin	List Biological Laboratories	Cat#: 181
Myelin oligodendrocyte glycoprotein peptide 35–55	GL Biochem	NA
Incomplete Freund’s Adjuvant (IFA)	Sigma-Aldrich	Cat#: F5506
Complete Freund’s Adjuvant (CFA)	Sigma-Aldrich	Cat#: F5881
Mitomycin C	Sigma-Aldrich	Cat#: 50-07-7
Ionomycin	Sigma-Aldrich	Cat#: I0634
Fixation/Permeabilization Concentrate	eBioscience	Cat#: 00-5123-43
Permeabilization Buffer (10X)	eBioscience	Cat#: 00-8333-56
PMA	Sigma-Aldrich	Cat#: 16561-29-8
Cytofix/Cytoperm solution	BD Bioscience	Cat#: 554714
Critical commercial assays
CD4+ T Cell Isolation Kits	Miltenyi Biotec	Cat#: 130-104-454
Pan Dendritic Cell Isolation Kit	Miltenyi Biotec	Cat#: 130-100-875
Mouse IL-17A ELISA MAX™ Deluxe Sets	BioLegend	Cat#: 432504
Mouse IL-10 ELISA MAX™ Deluxe Sets	BioLegend	Cat#: 431414
Mouse IFN-g ELISA MAX™ Deluxe Sets	BioLegend	Cat#: 430804
Mouse IL-1b ELISA MAX™ Deluxe Sets	BioLegend	Cat#: 432604
Mouse TNF-a ELISA MAX™ Deluxe Sets	BioLegend	Cat#: 430904
Mouse TGF-b ELISA LEGEND MAX™	BioLegend	Cat#: 433007
Mouse IL-23 ELISA MAX™ Deluxe Sets	BioLegend	Cat#: 433704
Mouse IL-21 ELISA LEGEND MAX™	BioLegend	Cat#: 446107
Mouse IL-6 ELISA MAX™ Deluxe Sets	BioLegend	Cat#: 431315
Experimental models: Organisms/strains
Cblb/mice	Dr. Josef M. Penninger	N/A
2D2 TCR mice	Jackson Laboratory	RRID: IMSR_JAX:006912
Card9–/– mice	Jackson Laboratory	RRID: IMSR_JAX:028652
Clec7a–/– mice	Jackson Laboratory	RRID: IMSR_JAX:012337
LysM Cre mice	Jackson Laboratory	RRID: IMSR_JAX:004781
Cd4 Cre mice	Jackson Laboratory	RRID: IMSR_JAX:017336
Rag1–/– mice	Jackson Laboratory	RRID: IMSR_JAX:002216
Cd11c Cre mice	Jackson Laboratory	RRID: IMSR_JAX:008068
CblbC373A mice	(Oksvold et al., 2008)	N/A
Clec4n–/– mice	Dr. Yoichiro Iwakura	N/A
Cblb−/−Rag1−/− mice	Mice crossed in our facility	N/A
Cblb−/−Clec4n−/− mice	Mice crossed in our facility	N/A
Cblb−/−Clec7a−/− mice	Mice crossed in our facility	N/A
Cblbf/f mice	Generated in-house	N/A
Cd4 Cre-Cblbf/fmice	Mice crossed in our facility	N/A
LysM Cre-Cblbf/f mice	Mice crossed in our facility	N/A
Cd11c Cre-Cblbf/f mice	Mice crossed in our facility	N/A
Oligonucleotides
Primer RNA:5′-CCCGGGCAAGGCTCAGCCATGCTCCTG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GCGGCCGCAATTCCCAGAGACATCCCTCC-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-CTAGGCCACAGAATTGAAAGATCT-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GTAGGTGGAAATTCTAGCATCATCC-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-CTGACAGGGAACAGAAGGTG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-TGCCTGCTTGCCGAATATC-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-AGGACTTTGCACTGGCGTAG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GCCAATGCTGCCGACTCCAG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GCTGTAACTTCTGAAGAAAAC-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GCGCGCCCCTCGAGCTAGAG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-TCTGGACTTGCCTCCTCTGT-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-CATTCCATCGCAAGACTCCT-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-TGGATGTGGAATGTGTGCGAG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-AAGGAGGGACTTGGAGGATG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GTCACTCACTGCTCCCCTGT-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-ACCGGTAATGCAGGCAAAT-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GCGGTCTGGCAGTAAAAACTATC-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GTGAAACAGCATTGCTGTCACTT-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-CTAGGCCACAGAATTGAAAGATCT-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GTAGGTGGAAATTCTAGCATCATCC-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-ACTTGGCAGCTGTCTCCAAG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GCGAACATCTTCAGGTTCTG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-CAAATGTTGCTTGTCTGGTG-3′	Integrated DNA Technologies	N/A
Primer RNA:5′-GTCAGTCGAGTGCACAGTTT-3′	Integrated DNA Technologies	N/A
Recombinant DNA
Accell Il6 siRNA	Dharmanon	Cat#: L-043739-00-0005
non-sense siRNA	Horizon Discovery	Cat#: D-001206-14-05
Software and algorithms
FlowJo	FlowJo, LLC and Illumina, Inc	https://www.flowjo.com/
GraphPad Prism 7.0	GraphPad Software, Inc.	https://www.graphpad.com/
SPSS software	IBM	https://www.ibm.com/analytics/spss-statistics-software
Image Lab Software	Bio-Red	https://www.bio-rad.com/
Adobe illustrator	Adobe	https://www.adobe.com/products/illustrator.html#