TNF promotes Th17 vaccine responses by enabling myeloid cell pattern recognition via Mincle

TNF promotes Th17 vaccine responses by enabling myeloid cell pattern recognition via Mincle | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TNF promotes Th17 vaccine responses by enabling myeloid cell pattern recognition via Mincle Robert Blamberg, Carl Haberkamp, Gabriel Kristian Pedersen, Ida Rosenkrands, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7495259/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Successful induction of protective T cells by subunit vaccines requires adjuvants. The adjuvant CAF01 potently induces robust Th17 responses that depend on the C-type lectin receptor Mincle and TNF. Mincle expression is low in resting macrophages, but upregulated by TNF. Here, we used conditional TNFR1-deficient mice to dissect cell type-specific contributions of TNF signaling to Th17 induction by the recombinant tuberculosis fusion protein H1 adjuvanted with CAF01. TNFR1 in myeloid cells was essential, whereas TNFR1 deletion in DC only partially reduced Th17 cells, and TNFR1 was not required in T cells. Constitutive, TNF-independent transgenic Mincle expression restored Th17 induction despite TNF blockade. Thus, regulation of Mincle by TNF plays a causal role, likely by controlling production of Th17-polarizing cytokines in monocytes. Together, we show that induction of Th17 by CAF01 requires TNF signaling in myeloid cells, with enhanced adjuvant sensing due to Mincle upregulation as a potential mechanism. Biological sciences/Cell biology Biological sciences/Immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction TNF is a key proinflammatory cytokine that contributes to various protective immune functions. It can signal via two TNF receptors: TNFR1, which is associated with pro-inflammatory functions, and TNFR2, which is involved in tissue regeneration and cell survival 1 . Besides its beneficial effects, TNF also plays a crucial role in many chronic inflammatory diseases like rheumatoid arthritis (RA), seronegative spondyloarthropathies, and inflammatory bowel disease (IBD), where a standard treatment strategy involves TNF blockade by antibodies (e.g. Infliximab or Adalimumab) or the human TNFR2-Fc fusion protein Etanercept 2 . Unwanted side effects of TNF blockade include increased susceptibility to fungal and bacterial infections, including reactivation of latent tuberculosis 3 . In addition, several studies reported an impaired immune response to vaccination due to TNF blockade, including reduced antibody responses to T cell-dependent vaccines for influenza 4 – 6 and hepatitis B 7–9 as well as for T cell-independent pneumococcal polysaccharide vaccines 9 , 10 . Subunit vaccines consist of highly purified, often recombinant, antigens that can be processed and presented by antigen-presenting cells (APC) to T cells (signal 1). Their immunogenicity for T cells depends on adjuvants that increase costimulatory molecule expression (signal 2) and production of cytokines to promote and modulate Th cell differentiation (signal 3). Aluminium salts have been used as adjuvants in humans for nearly a century to induce antibody responses, but fail to generate strong T cell responses that are required to protect from intracellular pathogens 11 . Complete Freund’s adjuvant (CFA), an emulsion of heat-killed Mycobacterium tuberculosis (MTB) in mineral oil, has been used in experimental animals for many decades to induce strong cellular immune responses, including T helper (Th)1 and Th17 cells, but its inflammatory side effects preclude its use in humans 12 . Inducing pathogen-specific Th17, along with Th1 cells, can be beneficial for protection against various intra- and extracellular bacteria and fungi. For example, to clear the extracellular bacterium Klebsiella pneumoniae in mice, IL-17 is essential to induce neutrophil recruitment 13 . IL-17 activates macrophage killing of Bordetella pertussis and plays a role in the successful vaccination with whole-cell pertussis vaccines 14 . Besides the recruitment of innate immune cells and the induction of other pro-inflammatory molecules, IL-17 promotes Th1 immunity. For example, it is critical to induce a Th1 response in Chlamydia muridarum- infected mice 15 , and in humans deficient in Th17 cells (due to a mutation in RORγt, the Th17 lineage-defining transcription factor 16 ), an impaired IFNγ response to mycobacteria was observed 17 . Further, induction of Th17 cells by the only licensed vaccine for tuberculosis, Mycobacterium bovis Bacille Calmette-Guérin (BCG), is required for protection after infection by recruiting Th1 cells 18 , 19 . The progress made in innate immune recognition of infectious danger signals through pattern recognition receptors (PRR) has fertilized the development of next-generation adjuvants that induce and shape both cellular and humoral immune responses 20 . One new adjuvant system named CAF01 (Cationic Adjuvant Formulation 01) induces a distinctive, mixed Th1/Th17 response, setting it apart from other next-generation adjuvants 11 . CAF01 consists of cationic N,N-dimethyl-N,N-dioctadecylammonium (DDA) liposomes, whose membrane contains the glycolipid trehalose6,6’-dibehenate (TDB) 21 . TDB is a synthetic, less toxic analog of trehalose-6,6’-dimycolate (TDM), an abundant and immunostimulatory mycobacterial cell wall glycolipid ( also known as cord factor). TDM and TDB bind to the activating PRR Mincle (macrophage-inducible C-type lectin) 22 , 23 . The Th17-inducing ability of CAF01 depends on the recognition of its component TDB by Mincle 23 and the resulting activation of the Syk-Card9 signaling pathway 24 . Consistent with the abundance of TDM in the mycobacterial cell wall, the Th17-inducing effect of Complete Freund’s adjuvant is strongly reduced in Mincle- and abrogated in Card9-deficient mice 25 . Mincle is expressed on myeloid cells, such as monocytes, macrophages and neutrophils, as well as on some dendritic cells (DC) 26 . Mincle is generally not expressed on adaptive immune cells, but some reports have found it under special circumstances on B cells 27 , 28 and T cells 29 . In contrast to other PRRs, Mincle is expressed at low levels on resting myeloid cells. In macrophages, stimulation with LPS 30 or the Mincle-ligands TDB and TDM upregulates Mincle expression, leading to enhanced activation and pro-inflammatory signaling 31 . We previously showed that TDB-/TDM-induced production of TNF was sufficient and essential for Mincle upregulation in macrophages, which was dependent on signaling via TNFR1 32 . The function of TNF in the induced expression of Mincle is conserved in human monocyte-derived macrophages after stimulation with BCG or LPS 33 . Upon vaccination in mice, genetic deletion of TNF or pharmacological blockade by Etanercept prevents CAF01-induced Th17 induction, but does not affect the IFNγ response 32 . While these findings established the essential function of TNF in CAF01-adjuvanted immunization, the cell type on which it acts to enable Th17 differentiation remains undefined. TNF receptors are expressed broadly in different tissues and immune cell types. We hypothesized that TNFR1 may be essential for Th17 induction in our vaccination model because of its pro-inflammatory function and its requirement for Mincle upregulation in macrophages. Conditional TNFR1 knockout mice crossed with cell-type-specific Cre-deleter strains provide a genetically defined system to dissect the pleiotropic effects of TNF 34 . These mice have been employed to elucidate that for protection against MTB infection, TNFR1 signaling in T cells is dispensable but essential in myeloid cells 35 . Here, we aimed to determine which immune cell type depends on TNF signaling to elicit both humoral and cellular immune responses following immunization with the recombinant MTB fusion protein H1 36 adjuvanted with CAF01. Hence, conditional TNFR1 knockout mice were generated using Cre-deleter strains specific for myeloid cells, dendritic cells (DC), or T cells. Furthermore, we employed Mincle-transgenic mice to investigate whether impaired vaccine responses during TNF blockade can be restored by re-establishing robust Mincle expression. Our results show that TNFR1 on myeloid cells is essential for inducing a Th17 response by H1/CAF01. TNFR1 on DC appears to contribute but is not essential, while TNFR1 on T cells is dispensable. Together, in the H1/CAF01 model, the lack of Mincle upregulation, probably on myeloid cells, can explain most of the effects of the TNF blocker. Results TNF blockade selectively prevents the Th17 but not the Th1 response to immunization We previously demonstrated that TNF is required for antigen-specific IL-17 production in mice vaccinated with the TB antigen H1, combined with the Mincle-dependent adjuvant CAF01 32 . Here, we immunized mice using the same antigen/adjuvant system while blocking TNF with the TNF blocker Etanercept (see Fig. 1a), restimulated splenocytes with H1 antigen and analyzed production of IL-17 and IFNγ by CD4 + T cells using intracellular cytokine staining (Fig. 1b). Consistent with our previous ELISA-based results, the frequency of antigen-specific Th17 cells was reduced by TNF blockade during immunization, whereas the H1-specific Th1 response was not affected (Fig. 1b; gating strategy shown in Fig. S1 ). We next sought to understand whether TNF is necessary to induce or to maintain antigen-specific Th17 cells. To address this question, we utilized Th17 fate reporter mice, in which IL-17A expression results in Cre recombinase-mediated constitutive YFP expression 37 . Indeed, while only few YFP + cells were detected in spleens of naive mice (Fig. 1c), CAF01/H1 induced a significant increase of YFP + cells, to a percentage comparable to the frequency of IL-17A + cells (0.3 vs. 0.5%). In mice immunized under TNF blockade, the number of YFP-marked cells decreased to the level found in unimmunized mice (Fig. 1c). In mice immunized without Etanercept, around 30% of YFP-marked cells produced IL-17 after H1 re-stimulation. The YFP-marked cells detected in mice treated with TNF blockade were unable to produce IL-17 after specific restimulation, comparable to unimmunized mice (Fig. 1d) Thus, we conclude that TNF is essential for inducing the differentiation of naïve T cells into Th17 cells in the H1/CAF01 vaccination model. Preventing TNF signaling via TNFR1 on myeloid cells reduces the local inflammatory response towards the adjuvant TNF affects multiple target cell types and exerts diverse biological effects. It signals through TNFR1 and TNFR2, with TNFR1 being more crucial for its pro-inflammatory function 1 . We aimed to determine which immune cell type requires TNFR1 signaling for the induction of Th17 cells. Considering TNFR1's vital role in upregulating the PRR Mincle on innate cells 32 , we hypothesized that TNF facilitates the recognition of the adjuvant by antigen-presenting and other innate immune cells. Alternatively, or in addition, an effect on B or T cells could explain the requirement for TNF in the H1/CAF01 vaccination model. We utilized cell type-specific deletion of TNFR1 to dissect the mechanistic cellular targets. We initially measured TNFR1 surface expression on immune cells from both immunized and unimmunized wild type (WT) mice (Fig. 2b and Fig. S3). We observed no significant TNFR1 expression on B cells. Consequently, we did not further pursue the role of TNFR1 signaling in B cells. TNFR1 was most highly expressed on neutrophils and classical monocytes, while Ly6C low monocytes expressed the receptor at a lower level and only on a subset (Fig. S3a). NK cells and NK T cells expressed TNFR1 at a level comparable to Ly6C low monocytes. cDC1 (CD88 - , CD26 + , CD64 - , MHCII + , CD11c + , XCR1 + ) and cDC2 (CD88 - , CD26 + , CD64 - , MHCII + , CD11c + , XCR1 - ) as well as T cells (CD4 T cells, CD8 T cells and γδ T cells) showed a lower expression level. On macrophages (Ly6G - , CD11b + , Ly6C low , F4/80 + ) only a small subpopulation showed a detectable TNFR1 expression. First, we checked the importance of TNF signaling via TNFR1 on myeloid cells. To this end, we crossed LysM-Cre mice 38 with mice in which exons 2–5 of the TNFR1 are floxed (Tnfrsf1a tm3.3Gkl , 34 ). This LysM-Cre-mediated deletion of the TNFR1 resulted in the complete loss of TNFR1 expression on neutrophils and a substantial reduction on Ly6C high inflammatory monocytes (Fig. 2b). On Ly6C low monocytes and on macrophages, TNFR1 was not reduced significantly by LysM-Cre mediated deletion. TNFR1 was expressed on subpopulations of Ly6C low monocytes and F4/80 + macrophages (Fig. S3a). Although LysM-Cre completely abolished TNFR1 staining in these subpopulations, MFI values for Ly6C low monocytes were not affected (Fig. 2b). In all other tested cell types, TNFR1 expression was not significantly changed, except for a slight increase on NK cells and a reduction on NK T cells. We will address the potential importance of TNFR1 on NK and NK T cells later. We first studied the effect of TNFR1 deficiency in myeloid cells on the local inflammatory response to immunization with H1/CAF01 (for the experimental layout see Fig. 2a). CAF01, a liposome-based adjuvant, forms a depot at the site of injection. Innate immune cells migrate to this location and slowly transport the adjuvant and antigen to the draining lymph node 39 . Upon s.c. immunization in the footpad, the measurement of its thickness over time can serve as a proxy for immune cell infiltration and inflammation at the site of injection. The robust footpad swelling after H1/CAF01 injection was reduced by approximately 50% in myeloid TNFR1 knockout (KO) mice compared to the control group (Fig. 2c), which corresponds well to the reduction observed in Tnf -/- mice 32 , indicating that TNF signaling in myeloid cells plays a significant role in the local inflammatory response. TNFR1 signaling in myeloid cells is essential for the antigen-specific Th17 response The absence of TNFR1 in myeloid cells completely prevented the induction of H1-specific production of IL-17 by LN cells, while the production of IFNγ and IL-10 remained unaltered (Fig. 2d, similar results observed for spleen cells see Fig. S4a). Of note, neither the floxing of TNFR1 alone (Fig. S5a) nor the insertion of the Cre recombinase under the LysM promoter (Fig. S5b) influenced the IL-17 production after immunization. The reduced IL-17 production was accompanied by the vaccine's inability to induce H1-specific Th17 cells in the myeloid TNFR1 KO mice, whereas the induction of Th1 cells was unaffected (Data from LN in Fig. 2e, comparable results were observed for spleen Fig. S4b, c). In summary, the absence of TNFR1 on myeloid cells abrogates the Th17 response induced by the Mincle-activating adjuvant CAF01, replicating the effect of genetic whole body deletion of the Tnf gene as we showed before 32 . TNF contributes to antibody induction, but TNFR1 on myeloid cells is dispensable We also tested the consequence of genetic deletion or pharmacologic blockade of TNF versus the specific knockout of TNFR1 in myeloid cells for the induction of the humoral immune response. To do so, we measured H1-specific antibody (Ab) titers after two injections of H1/CAF01 (Experimental layout Fig. S2a). Knockout of TNF reduced titers of all studied isotypes by more than one order of magnitude (Fig. S2b). It is well established that the formation of germinal centers is impaired in Tnf -/- mice 40 . Transient pharmacological TNF blockade of the response to vaccination by sequential treatment with Etanercept and a neutralizing anti-TNF antibody caused a weaker but significant reduction in H1-specific antibody (Ab) titers of the proinflammatory isotypes IgG2c and IgG2b, but not of IgG1 (Fig. S2b). In contrast to the effect of genetic deletion or pharmacological inhibition of TNF, the myeloid cell-specific TNFR1 KO did not affect the induction of H1-specific Ab after immunization (Fig. S2c). TNFR1 on DC plays a limited role in the antigen-specific Th17 response and is dispensable for induction of H1-specific antibodies After we found that the TNFR1 on myeloid cells is essential for the Th17 response, we wanted to test the importance of the TNFR1 on DCs. For this, we employed two well-characterized DC-targeting Cre-deleter mouse lines. First, a Cre knock-in at the Clec9a locus specifically deletes loxP -flanked genes in DC progenitors 41 . In Clec9a-Cre het ; TNFR1 fl/fl mice, we observed a complete absence of TNFR1 staining on cDC1 and a significant, but incomplete reduction (ca. 65%) of TNFR1 surface protein on cDC2 (Fig. 3a, representative histograms Fig. S6a). Besides DCs, there was a reduction of TNFR1 expression on macrophages and on NK cells, whilst the remaining tested cell types had intact TNFR1 expression. We will address the importance of NK cells on the induction of an antigen-specific Th17 response later. To rule out the possibility that the remaining TNFR1 expression on cDC2 leads to an incorrect conclusion, we used CD11c-Cre-mediated deletion 42 of TNFR1 in addition. In this model, TNFR1 staining was completely absent on cDC1 and cDC2 (Fig. 3b, representative histograms Fig. S6b). In contrast to the Clec9a-Cre model, there was also a reduction in TNFR1 surface protein across all T cell subsets. Additionally, a significant reduction of TNFR1 was observed in Ly6C low monocytes and in macrophages. In both models for deleting TNFR1 on DC, the inflammatory response at the site of injection, measured by the footpad swelling, was unaffected (Fig. 3c, d). Clec9a-mediated TNFR1 deletion did not alter cytokine production after the restimulation of LN cells from immunized mice (Fig. 3e). These findings were confirmed by the unaltered induction of Ag-specific Th17 and Th1 cells after immunization (Fig. 3f). This suggests that TNFR1 expression on cDC1 cells is not required for H1/CAF01 induced Th17 responses. The CD11c-Cre-mediated complete TNFR1 deletion on all DC subsets did not affect the production of IFNγ and IL-10 in LN cells (Fig. 3g) nor the induction of Th1 cells (Fig. 3h). In contrast, it led to a significant reduction in IL-17 production upon restimulation of LN cells with H1, with 75–80% reduction when measured by ELISA (Fig. 3g), or around 50% reduction in LN CD4 + T cells producing IL-17A, as measured by intracellular cytokine staining (Fig. 3h).The same pattern was observed after restimulation of spleen cells and measurement by ELISA (Fig. S8a, c) and after intracellular cytokine staining of spleen cells (Fig. S8b, d). No significant inhibition of IL-17 production was observed in CD11c-Cre + ; TNFR1 wt/wt mice (Fig. S5c). In summary, TNFR1 on DC seems to play a role in inducing a Th17 response after H1/CAF01 immunization, but the Th17 response does not entirely depend on it, as observed for the TNFR1 on myeloid cells. We also measured the H1-specific Ab titer in H1/CAF01-immunized CLec9a-Cre het ; TNFR1 fl/fl and CD11c-Cre + ; TNFR1 fl/fl mice. In both models, the specific Ab titers were similar to those in the control group (Fig. S2c). This implies that TNFR1 on DC is dispensable for the H1/CAF01-induced Ab response. TNFR1 on T cells is not required for the antigen-specific Th17 response The impaired Th17 response in CD11c-Cre + ; TNFR1 fl/fl mice was associated with a reduction in TNFR1 surface protein on T cells (Fig. 3b), raising the question whether TNFR1 on T cells is required for Th17 differentiation in our immunization model. To address this, we used the Lck-Cre mouse 43 to delete the floxed TNFR1 during thymic development of T cells. In this model, TNFR1 was no longer present on the surface of CD4 + and CD8 + T cells as well as on γδ T cells and NK T cells (Fig. 4a, representative example see Fig. S7). Besides this, TNFR1 expression on all other studied cell types was unaffected. Thus, Lck-Cre-mediated deletion was efficient and specific in the T cell compartment. Of note, a lower frequency of CD4 + T cells was observed in naïve and immunized Lck-Cre + ; TNFR1 fl/fl mice (Fig. 4b). The local inflammatory response in the footpad remained unchanged (Fig. 4c). The absence of TNFR1 did not significantly affect IL-17 production following the restimulation of LN cells from immunized mice (Fig. 4d, comparable to spleen cells Fig. S9a). However, a significant reduction in IFNγ and IL-10 production was noted. The frequency of induced specific Th17 and Th1 cells in the LN remained unchanged (Fig. 4e, spleen comparable see Fig. S9b). In conclusion, the TNFR1 on T cells may contribute to their overall numbers or function, but is not essential for the induction of specific Th17 cells by the H1/CAF01 vaccine. The adjuvant CAF01 induced Th17-polarizing cytokines in monocytes in a TNF-dependent manner. We previously found that CCR2 + monocytes, but not neutrophils, are critical for IL-17 induction by CAF01 44 . Therefore, we focused on monocytes isolated from mouse bone marrow cells. We observed, that there was a TNF-dependent transcriptional upregulation of Mincle after stimulation with the Mincle ligand TDB (contained in CAF01) as well as with the mycobacterial cord factor TDM (Fig. 5a). The expression of IL-6 and IL-1β was TNF-dependently upregulated by both Mincle ligands (Fig. 5b, c, d). In summary, we show that the CAF01 constituent TDB induced the Th17 polarizing cytokines IL-6 and Il-1β in monocytes, probably due to the TNF-dependent upregulation of Mincle. Transgenic constitutive expression of Mincle uncouples its surface expression from TNF signaling Next, we aimed to assess whether the impaired Mincle upregulation in mice treated with Etanercept or lacking TNFR1 in myeloid cells is causal in the abrogation of Th17 induction after vaccination. To achieve this, we used a transgenic mouse model in which Mincle is constitutively expressed at a high level (Mincle tg , 45 ). Additionally, to rule out confounding factors due to the regulation of endogenous Mincle by TNF signaling, the Mincle tg mice were crossed onto a Mincle-deficient background ( Clec4e -/ - , further referred to as Mincle -/- 46 ). To determine Mincle regulation in these mice, bone marrow-derived macrophages (BMDM) were generated and stimulated with the Mincle ligand TDM (Fig. 6a, representative Histogramms Fig. S10a). Mincle was expressed at low levels on resting WT BMDM and as expected strongly upregulated in a TNF-dependent manner after stimulation. In Mincle -/- ; Mincle tg BMDM, Mincle was highly expressed under resting conditions, and its expression level was unaffected by the TNF blocker Etanercept. TNF-independent, high constitutive Mincle expression can rescue Th17 induction We immunized the Mincle -/- ; Mincle tg mice to test the effect of TNF block when Mincle expression is no longer regulated (Experimental design Fig. 6b). The local inflammatory response, measured by footpad swelling, was reduced in WT mice due to TNF block (Fig. 6c). The magnitude of this reduction was comparable to that seen in mice lacking TNFR1 on myeloid cells. In the transgenic mice, footpad swelling was greater compared to WT mice. TNF blockade with Etanercept reduced it, but only to the level seen in non-TNF-blocked WT mice. IL-17 production after restimulation of LN cells from immunized mice was, as expected, entirely prevented in TNF-blocked WT mice (Fig. 6d, comparable results for spleen cells, see Fig. S10b). In contrast, IL-17 production was restored in TNF-blocked transgenic mice to the level observed in immunized WT mice. The production of IFNγ and IL-10 was not altered due to the TNF block. Correspondingly, the frequency of specific Th17 cells was reduced in immunized TNF-blocked WT mice, whereas in Mincle-transgenic mice, the TNF blocker did not affect the frequency of Th17 cells compared to immunized WT mice (Fig. 6e, comparable results for spleen cells, see Fig. S10c, d). Under physiological conditions, Mincle is expressed on myeloid cells and some dendritic cells (DC) 26 . Some reports have found it under special circumstances on B cells 27 , 28 and T cells 29 . We now tested the expression of the transgenic mincle on celltypes which are not known to express mincle in WT mice. We found that, NK and NK T cells from Mincle -/- ; Mincle tg mice expressed significant Mincle protein on the surface, in contrast to their WT counterparts (Fig. S11a). This observation raised the question whether NK and NK T cells contributed to the observed rescue of Th17 induction. Therefore, we tested whether NK1.1-positive cells contribute critically during immunization in WT and transgenic mice under TNF blockade by repeatedly injecting the anti-NK1.1 antibody PK136 (see experimental scheme in Fig. S11b), resulting in the complete depletion of NK1.1-positive cells (Fig. S11c). The immunization of WT mice in the absence of NK1.1-positive cells still led to IL-17 induction (Fig. S11d), with levels comparable to those observed in previous experiments. Also, the complete prevention of specific IL-17 production due to TNF block during immunization did not depend on NK1.1-positive cells. Therefore, we can exclude a potential impact of the observed differences in TNFR1 expression on NK and NK T cells after Clec9a-Cre-mediated deletion (Fig. 3a) or CD11c-Cre-mediated deletion (Fig. 3b). In addition, the rescue of the IL-17 production in TNF-blocked Mincle -/- ; Mincle tg mice was unaffected by the absence of NK1.1-positive cells (Fig. S11d). Together, the Th17 rescue effect observed in the transgenic mice most likely originates from the constitutive expression of Mincle on either monocytes or DC, since NK1.1-positive cells were dispensable and transgenic Mincle was not stably expressed on T and B cells (Fig. S11a). Discussion We employed conditional TNFR1-deficient mice to dissect the mechanism underlying our previous observation that TNF is required for Th17 induction by the Mincle-dependent adjuvant CAF01 32 . The complete loss of IL-17-producing Th cells in mice lacking TNFR1 in myeloid cells reproduced the effect of whole-body TNF deficiency and confirmed the selective loss of Th17, but not Th1, differentiation. Although TNFR1 is highly expressed in monocytes and in neutrophils, our previous demonstration that CAF01-induced Th1/Th17 induction requires CCR2 + monocytes, but not Ly6G + neutrophils 44 , allows us to conclude that only TNFR1 on monocytes or macrophages is essential for Th17 induction. TNFR1 deletion in DC by CD11c-Cre resulted in a significant but incomplete reduction in IL-17-producing CD4 + T cells, whereas Clec9a-Cre-mediated deletion had no effect on Th cell differentiation at all. Finally, deletion of TNFR1 in T cells did not impair Th17 induction at all. Together, the use of cell type-specific TNFR1 knockout mouse lines clearly revealed that monocytes/macrophages are the critical target cells for promoting Th17 induction by TNF. Mechanistically, the partial restoration of Th17 adjuvanticity in Mincle −/− ; Mincle tg mice undergoing TNF blockade indicates that downregulation of Mincle expression contributes substantially to the loss of Th17 differentiation by impairing myeloid cell sensing of the adjuvant TDB. In contrast to the Th17 response, and consistent with our previous finding that IFNγ responses were unaffected by TNF 32 , we found that Th1 differentiation is independent of TNF. In this context, it is of interest that DDA liposomes, in which the Mincle ligand TDB would be incorporated to obtain CAF01, possess an intrinsic, moderate IFNγ- but not IL-17-inducing adjuvant activity, which is independent of Mincle 23 . Therefore, the Mincle-dependent adjuvant activity of CAF01 is more crucial for inducing the Th17 response but not the Th1 response. Activation and priming of naïve T cells require MHC-restricted antigen recognition by the TCR (signal 1) in conjunction with costimulation (signal 2). TNF and TNFR1 signaling appear not to be required for T cell priming in vivo by the recombinant H1/CAF01 vaccination, as the induction of antigen-specific Th1 cells and the secretion of IFNγ and IL-10 upon restimulation in vitro were not affected by genetic deficiency or pharmacological blockade. However, the differentiation of Th cells is controlled by the cytokine environment in the draining lymph node and the spleen (signal 3). IL-1β, IL-6, IL-23 and TGFβ promote Th17 differentiation, while type 1 interferons, IFNγ, and IL-4 inhibit it 47 . We previously showed that IL-1 receptor signaling via Myd88 is required for the Th17-inducing effect of CAF01 in vivo 48 . Here, we found that the TNF blocker Etanercept strongly impaired the expression of IL-6 and IL-1β in monocytes stimulated with the Mincle ligand TDB in vitro . This reduction in Th17-inducing cytokines complements the in vivo results showing that TNFR1-deficiency in myeloid cells abrogates Th17 induction, and supports the interpretation that TNFR1 signaling drives the activation of monocytes and macrophages that generate signal 3. In contrast, TNFR1 signaling in DC seems less important for Th17 differentiation, with intact Th17 responses after complete TNFR1 deletion on cDC1s, and a reduced capacity to produce IL-17 following CD11c-Cre-mediated complete deletion of the TNFR1 on all DC subsets. Together, the results from using LysM-Cre, Clec9a-Cre and CD11c-Cre deleter mice suggest that antigen presentation by cDC is not regulated by TNF, whereas signal 3 is derived mainly from monocytes/macrophages, and perhaps cDC2, activated by the Mincle-dependent adjuvant and depends on TNFR1 signaling. While the specificity of Cre-mediated TNFR1 deletion in different immune cells was overall according to our expectations, the distinction between monocytes, macrophages and different DC subsets using the existing markers is not absolute and subject to adjustments as new insights are published. In relation to our results, the description of the DC3 lineage by Ginhoux and colleagues 49 as Th17-inducing cells derived from Ly6C + monocyte-DC precursors is of special interest because fate mapping in LysM-Cre-R26TdT mice showed a high proportion of marked DC3. It is therefore possible that the myeloid cell populations required for Th17 induction, which we considered as monocytes/macrophages, may also contain DC3. It was reported before, that CD11c-Cre shows leaky expression in different hematopoietic lineages, that can lead to deletion of sensitive floxed alleles 50 . We observed a partial deletion of TNFR1 on macrophages and Ly6Clow monocytes by CD11c-Cre, therefore it is also possible that the observed reduction in Th17 cells is not caused by the deletion of TNFR1 in cDCs, and that the cytokines secreted by TNFR1-activated macrophages and Ly6Clow monocytes are critical for Th17 differentiation. Based on reports showing that TNF block can reduce Th17 and Th1 cell differentiation after anti-CD3/CD28-induced differentiation of naïve human T cells in vitro 51 , 52 , we considered that TNF may act directly on CD4 + T cells to promote Th17 differentiation and therefore included the Lck-Cre deleter strain. TNFR1 surface protein levels were relatively modest in T cells and we found no evidence for the importance of the TNFR1 on T cells in Th17 induction. However, we note that the effects of TNF on T cells can depend on signaling through TNFR2, which we did not address here. First, it was described in human T cells that TNF signaling via TNFR2 but not TNFR1 acts as a costimulatory signal for T cell receptor activation 53 . In addition, a recent publication revealed that TNF signaling through TNFR2 together with TGFβ enhances Th17 induction 54 . In the same paper, it was demonstrated that IL-1β induces TNF in T cells, and that, in contrast to IL-6, TNFR2 is essential for the IL-1β effect on Th17 differentiation. Clearly, the potential involvement of TNFR2 in regulation of vaccination-induced Th cell differentiation deserves further investigation. Besides the direct effects on T cells, it was shown in rheumatoid arthritis (RA) patients that TNF induces IL-6 in monocytes, via TNFR1 and TNFR2, which leads to an increase in Th17 cells 55 . This reflects our finding that Etanercept prevents the induction of IL-6 in murine monocytes. Another paper described that TNF promotes differentiation of human monocytes into DC, which in turn have the capacity to induce Th17 responses 56 . Alternatively, an indirect effect was described in RA patients treated with the TNF blocker Adalimumab (but not observed for Etanercept) that regulatory T cells increase, which inhibit IL-6 production in monocytes via IL-10 57 . We did not find increased IL-10 levels in mice after TNF blockage by Etanercept, arguing that this mechanism may be specific to RA or humans, or that the type of TNF blocker makes a difference. In addition to the Th cell response, we analyzed whether TNF/TNFR1 signaling contributes to the generation of specific antibodies after H1/CAF01 immunization. It is known that the knockout of TNF in mice prevents germinal center (GC) formation, disrupts follicular DC networks and consequently leads to impaired antibody responses 58 . Even transient blockade of TNF by Etanercept can have an adverse effect on GC organization and reduce the number of follicular DC 59 . TNFR1 contributes to GC formation through its role in FDC differentiation 606162 . In the H1/CAF01 immunization model, we found that induction of H1-specific antibodies was markedly reduced in whole body TNF knockout mice and blockade of TNF during the immunization period was moderately inhibitory. In contrast, TNFR1 deletion in myeloid cells and in cDC did not affect antibody titers. Thus, the loss of antigen-specific Th17 cells observed in the myeloid TNFR1 knockout mice was not linked to antibody responses, indicating that, although Th17 cells can act in vitro as B cell helpers and drive an isotype switch towards IgG2a and IgG3 63 , the IgG2a response to H1/CAF01 is likely driven by Th1 cells 64 . We previously linked the requirement of TNF for the induction of an IL-17 response after immunization with the Mincle-dependent adjuvant CAF01 to upregulation of this PRR 32 . We have now found that TNFR1 signaling on myeloid cells is essential for Th17 induction but dispensable for Th1 or humoral responses. We therefore postulate that the missing upregulation of Mincle after binding the adjuvant interrupts a positive TNF-dependent feed-forward loop, thwarting myeloid cell activation and secretion of Th17-polarizing cytokines. It was found that in Mincle KO mice, the induction of IL-6, IL-1β, and IL-23 by CFA was reduced, concomitant with a strongly reduced Th17 response 25 . In line with our model, we found that after stimulation of monocytes with the CAF01 component TDB, the increase in Mincle expression was prevented by blocking TNF, which was accompanied by the prevention of the TDB-induced expression of IL-6 and IL-1β. Significantly, constitutive transgenic Mincle expression prevented the loss of the specific Th17 response caused by the TNF blocker, further supporting the notion that disrupted upregulation of Mincle in myeloid cells impairs the sensing of the TDB adjuvant. The Mincle-transgenic mouse line was used before to study the role of Mincle signaling in pneumonia caused by pneumococci and by Staphylococcus aureus 65 , 66 . A limitation of this mouse model is the ubiquitous expression of transgenic Mincle mRNA that contrasts the specific expression of endogenous Mincle in myeloid cells. However, surface localization of Mincle protein requires the presence of the adapter protein Fc receptor gamma chain (encoded by Fcer1g ) that interacts with a conserved arginine residue in the transmembrane domain of Mincle 67 . Fc receptor gamma is expressed in a more restricted pattern on myeloid cells, mast cells, and innate lymphoid cells, including NK and NKT cells. Indeed, in addition to myeloid cells and some DC, we found robust transgenic Mincle protein expression on NK1.1 + cells, but these were not contributing to Th17 induction as shown by antibody-mediated depletion experiments. In summary, we found that Mincle upregulation is a mechanism through which TNF enhances adjuvant recognition and responsiveness. This process is crucial in myeloid cells, especially in monocytes, where the lack of TNFR1 signaling prevents the secretion of Th17-promoting cytokines. Besides the Mincle-dependent adjuvant CAF01, the effect of TNF on Mincle regulation on myeloid cells could also be relevant for other, clinically used vaccines. The widely used aluminum salt-based adjuvants stimulate the immune system by inducing cell death 68 . Mincle binds molecules released by dying cells, such as spliceosome-associated protein 130 (SAP130) 67 and β-glucosylceramide 69 . Therefore, Mincle and its regulation by TNF may be essential for recognizing these aluminium adjuvant-induced DAMPs. In addition, the pneumococcal antigen glycolipid glucosyl-diacylglycerol (Glc-DAG) was identified to bind to Mincle 66 , 70 . Whether Glc-DAG is present in pneumococcal polysaccharide vaccine preparations and acts as a built-in Mincle-dependent adjuvant remains to be determined. If so, the reduced immunogenicity of the pneumococcal polysaccharide vaccine 9 , 10 in patients receiving TNF blockers could be caused by a similar mechanism. Therefore, further studies on the role of Mincle in the adverse effects of TNF blockade on response to different vaccine adjuvants are necessary. This knowledge will be valuable in gaining a fundamental understanding of the mechanisms of action, which are essential for the further design and application of vaccines and adjuvants. Methods Mice All mice were bred and housed under specific pathogen-free conditions at the Präklinische Experimentelle Tierzentrum of the Medical Faculty in Erlangen, Germany, or at the Statens Serum Institute, Denmark. Frozen sperm cells of TNFR1 fl/wt mice 34 were obtained from the European Mouse Mutant Archive in Athens, Greece (EMMA strain 11019, B6.129P2(Cg)-Tnfrsf1a tm3.3Gkl/Flmg , archived on a C57BL/6 background), and used to re-derive the mice by in vitro fertilization at the Transgenic Facility of the Friedrich-Alexander-University Erlangen-Nürnberg. To generate cell type-specific conditional TNFR1 KO lines, TNFR1 fl/fl mice were crossed with LysM-Cre (B6.Lyz2 tm1(cre)Ifo ; 38 ), Clec9a-Cre B6J.B6N(Cg)-Clec9atm2.1(icre)Crs/J; 41 ), CD11c-Cre (B6.Cg-Tg(Itgax-cre)1-1Reiz/J; 42 ), or Lck-Cre (Tg(Lck-cre)1Jtak 43 ) mice. Mincle tg mice 45 were obtained from Sho Yamasaki and crossed with Mincle KO mice (Clec4e tm1.1Cfg ) generated and provided by the Consortium for Functional Glycomics 46 . Tnf −/− mice 40 were provided by Dr. Ulrike Schleicher. C57BL/6N mice were purchased from Charles River Laboratories. At the end of the experiment the mice were euthanized by cervical dislocation. All mouse experiments were approved by the Regierung von Unterfranken (protocol number 55.2.2-2532-2-1641). IL-17A fate reporter mice were made by crossing the Il17a tm1.1(icre)Stck /J (IL-17cre) strain with the B6.129X1-Gt(ROSA)26Sor tm1(EYFP)Cos /J (R26R-EYFP) strain 37 (from The Jackson Laboratory (Bar Harbor, USA)) and handled at the experimental animal facility at Statens Serum Institut. At the end of the experiment the mice were euthanize by cervical dislocation. Experimental work was conducted in accordance with the regulations of the Danish Ministry of Justice and the Danish National Experiment Inspectorate under permit 2017-15-0201-01363 and in compliance with the European Community Directive 2010/63 EU for the care and use of laboratory animals. Immunizations To study the cellular immune response, we used a 7-day immunization protocol, as previously described 24 , 32 , 71 . Mice were immunized s.c. in both footpads with 50 µl CAF01 21 mixed with 1 µg H1 36 per foot, except in the IL-17A fate-reporter mice, where s.c. immunization was performed at the base of tail using a 100µl volume. Unimmunized mice were injected with the same volume of PBS. All footpad immunizations were performed on unconscious animals after inhalation anesthesia with 4% Isoflurane. For the immunization in the base of tail (s.c.) no anesthesia was used. After footpad immunization, the footpad thickness was measured before the immunization (day 0) and every second day after immunization. The footpad swelling was calculated by subtracting the initial thickness (on day 0) from the later obtained values. On day 7 after immunization, the mice were killed, and the inguinal and popliteal lymph nodes as well as the spleen were isolated. For the experiments requiring TNF blockade, Etanercept (3 mg/kg in 100 µl PBS) was injected s.c. in the flank (without anesthetization) on the day of immunization and every other day thereafter. The control group was injected in parallel with 100 µl PBS. For the depletion of NK1.1-positive cells, an anti-NK1.1 antibody (clone PK136, Leinco Technologies) was used in addition to etanercept treatment in the 7-day immunization protocol. 250 µg/mouse of this antibody or an isotype control (clone C1.18.4, Leinco Technologies) was injected i.p. (without anesthetization) one day before and a second time two days after immunization with H1/CAF01. To study the humoral immune response, a 5-week immunization protocol was used. Here, the mice received a second immunization at day 21 (2 µg H1 in 100 µl CAF01, s.c. base of tail). Blood was collected at day 0 (no H1-specific Ab detectable, data not displayed) and after the killing on day 36. To assess the role of TNF on the Ab production, additionally etanercept (3 mg/kg in 100 µl PBS) was injected s.c. in the flank on the day of immunization and every other day until day 18. Then, to prevent hindrance of the TNF block by the formation of anti-etanercept Ab, we switched to injecting anti-murine TNF Ab (200 µg/mouse, clone TN3-19.12, Leinco Technologies) every second day starting from day 20. A control group was injected with a matching isotype control (clone PIP, Leinco Technologies). All s.c. and i.p. injections of antibodies and etanercept were performed without anesthetization. In this protocol, the mice were killed 1 week after the booster immunization and blood was collected. Differentiation and stimulation of BMDM Bone marrow cells were isolated from C57BL/6 mice and Mincle −/− ; Mincle tg mice and differentiated as described before 32 . TDM was dissolved in isopropanol and coated to the bottom of the well (final concentration of 2 µg/ml) by allowing the isopropanol to evaporate. For the control wells, the same volume of isopropanol was evaporated. The BMDM were seeded in a flat-bottom 96-well plate (2*10 5 cells/well). Etanercept 100 µg/ml was added to the respective wells. The cells were stimulated for 24 h. Isolation and stimulation of BM monocytes To isolate monocytes from the bone marrow, the monocyte Isolation Kit (Miltenyi Biotec, Order No.: 130-100-629) was used according to the instructions of the producer. BM monocytes were stimulated by seeding them into a flat-bottom 96-well plate (2*10 5 cells/well). The wells were coded before with isopropanol, TDB (5 µg/ml), or TDM (2 µg/ml) as described above. Etanercept (final concentration 100 µg/ml) was added to the respective wells. The cells were stimulated for 24 h. Restimulation of cells from spleen and lymph node A single cell suspension of lymph node cells was obtained by mechanically dissociating the LN using a 70 µm cell strainer. The spleen was also mashed through a 70 µm strainer, and additionally, the erythrocytes were lysed using ammonium chloride. 5 x 10 5 LN and spleen cells were restimulated in a 96-well U-bottom cell culture plate for 96h with H1 (1 µg/ml). Cytokine ELISA Cytokines were measured in the supernatants of restimulated cells. For this purpose, the following ELISA kits were used according to the manufacturer's instructions: IFNγ (R&D Systems, DY485), IL-17 (R&D Systems, DY421), IL-10 (R&D Systems, DY417), and IL-6 (R&D Systems, DY406). Flow cytometry After blocking with anti-mouse CD16/32 antibody (eBioscience) the dead cells were stained with the Fixable Viability Dye eFluor 506 (Invitrogen 65-0866-14). Next, fluorophore-coupled antibodies were used to stain for the surface markers (see Table S1 for a list of the used antibodies). For the staining of Mincle, an uncoupled primary antibody (MBL, clone 4A9) was used and detected by using a suitable fluorescent-labeled secondary antibody. For intracellular cytokine staining, 1.5 x 10 6 cells were restimulated in a 96-well plate using 5 µg/ml of each of the three H1 peptides Ag85B p241−255 , Ag85B p261−280 , ESAT-6 p1−15 (obtained from Peptides and Elephants) or using the whole H1 protein. The cells were stimulated for one hour, followed by five hours in the presence of Brefeldin (10 µg/ml). After surface staining and fixation with 1% PFA overnight at 4 o C, the cells were permeabilized by saponin, and antibodies for the intracellular staining were added. Flow cytometry measurements were performed using an LSRFortessa (BD Bioscience, 5-laser configuration, Model No. 647794E6). The flow cytometry data were analyzed using FlowJo (BD Live Sciences, v. 10.7.1). We defined the cell types by the following marker (gating strategy in Fig S3): B cells (CD19 + , CD3-), neutrophils (CD3 − , CD19 − , NK1.1 − , Ly6C + , Ly6G + ), classical monocytes (CD3 − , CD9 − , NK1.1 − , Ly6G − , CD11b + , Ly6C high ), Ly6C low monocytes (CD3 − , CD19 − , NK1.1 − , Ly6G − , CD11b + , Ly6C low ), NK cells (CD3 − , CD19 − , NK1.1 + ), NK T cells (CD3 + , CD8 − , CD4 − , NK1.1 + ), macrophages (CD3 − , CD9 − , NK1.1 − , Ly6G − , CD11b + , Ly6C low , F4/80 + ), cDC1 (CD3 − , CD19 − , NK1.1 − , CD88 − , CD26 + , CD64 − , MHCII + , CD11c + , XCR1 + ), cDC2 (CD3 − , CD19 − , NK1.1 − , CD88 − , CD26 + , CD64 − , MHCII + , CD11c + , XCR1 − ) CD4 T cells (CD3 + , CD19 − , γδ TCR − , CD4 + , CD8 − ), CD8 T cells (CD3 + , CD19 − , γδ TCR − , CD8 + , CD4 − ), γδ T cells (CD3 + , CD19 − , γδ TCR + ) Th17 cell (CD3 + , CD4 + , CD8 − , IFNγ − , IL-17 + ), Th1 cell (CD3 + , CD4 + , CD8 − , IFNγ + , IL-17 − ). RNA isolation and qPCR Cells were lysed using Tri reagent (Bio&Sell, order No: BS67.211.0100). The RNA was isolated using the Direct-zol™ -96 RNA isolation Kit (ZymoResearch, cat. No.: R2056) according to the instructions of the producer. The RNA was transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat No.: 4368814). To perform the qPCR, primers and probes from the KiCqStart® Probe Assay (Sigma-Aldrich) were used for Clec4e (Mincle) and Hprt (sequence see Table S2). For Il6 and Il1b , the primers were used together with a probe from the Roche UPL universal probe library (sequence see Table S2). Gene expression was normalized using the Hprt expression, and the fold change was calculated using the 2 −ΔΔct method 72 , with isopropanol-treated samples serving as calibrators. Serum collection and H1-specific Ab ELISA Blood was collected at the end of the experiment by retroorbital bleeding from unconscious animals after i.p. application of Ketamin (100 µg/g bodyweight) together with Xylazin (10 µg/g bodyweight). Afterwards, still under narcosis, the mice were killed by cervical dislocation. Sera were separated from the blood by centrifugation (900 g, 10 min). H1 (400 ng/ml) was adsorbed overnight onto an ELISA plate (Sarstedt, ref: 82.1581.200). The non-bound H1 was discarded, and after BSA block (1% BSA in PBS), the sera were applied at different dilutions. The sera were washed away and the bound H1-specific Abs were detected using the following HRP-coupled secondary Ab: goat anti-mouse IgG1 (Southern Biotech, 1071-05, 1:4000), goat anti-mouse IgG2c (Southern Biotech, 1078-05, 1:4000), goat anti-mouse IgG2b (Southern Biotech, 1091-05, 1:4000). Statistics For statistical analysis, GraphPad Prism (GraphPad Software, LLC, version 9.5.1) was used. The used statistical test for every graph is indicated in the figure legends. In all graphs, each dot corresponds to one mouse, the height of the bar indicates the mean. For the two-way ANOVA tests the normal distribution of the data was tested using the Shapiro-Wilk test. If the requirement of the normal distribution was not meet, the ANOVA was performed on log transformed values, this is indicated in the figure legend. The analysis of Ab titers was only done for immunized groups. To this end, a correlation was modeled between the measured OD values and the respective dilution step. For this, a three-parameter non-linear regression with the equation OD = Bottom + (Top-Bottom)/(titer/mid-titer point) was used. The mid-titer point was obtained together with its SEM, and to compare these values between different groups, a one-way ANOVA followed by Dunnett’s multiple comparison test was performed (or Mann-Whitney U test in case of two groups). The p-values of all tests are displayed on the respective comparisons; to maintain clarity, only meaningful comparisons are shown in the graphs. Declarations Data availability All data generated or analysed during this study are included in this published article and its supplementary information files. Acknowledgements We thank Drs. Lars Nitschke, Kai Hildner, and David Vöhringer for providing different Cre-deleter mouse lines. We thank Drs. Gerhard Krönke and Jürgen Rech for supplying Etanercept. Many thanks to Dr. Dieter Engelkamp and Martina Döhler for IVF of TNFR1fl mice. Animal husbandry by Manfred Kirsch and the PETZ staff is highly appreciated. Many thanks to Barbara Bodendorfer for her technical support. The work was done as part of the doctoral thesis of R. B.. Funding This work was supported by Deutsche Forschungsgemeinschaft grants RTG 2599 “Fine Tuners of the Adaptive Immune Response” and LA 1262/8-1 to R.L. . U.S. was supported by RTG 2740 "ImmunoMicroTope" project A6. The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Author contributions R.B. performed all experiments, except the IL-17 fate-mapping mouse experiment, analysed and visualized the data, drafted and revised the manuscript. C.H. contributed preliminary analysis in Mincle tg mice. G.K.P. performed the IL-17 fate-mapping mouse experiment, discussed results, and revised the manuscript. I.R., U.S., B.U.S., S.Y. provided mice or critical reagents, discussed results, and revised the manuscript. R.L. designed and supervised the study and drafted and revised the manuscript. All authors read and approved the final manuscript. Disclosure and competing interest statement I.R. is coinventor on a patent of the cationic adjuvant formulations (CAF). All rights have been assigned to Statens Serum Institut, a Danish not-forprofit governmental institute. 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All rights have been assigned to Statens Serum Institut, a Danish not-forprofit governmental institute. All other authors declare no financial or non-financial competing interests. Supplementary Files SupplementarydataBlamberg.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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I.R. is coinventor on a patent of the cationic adjuvant formulations (CAF). All rights have been assigned to Statens Serum Institut, a Danish not-forprofit governmental institute. \nAll other authors declare no financial or non-financial competing interests.","formattedTitle":"TNF promotes Th17 vaccine responses by enabling myeloid cell pattern recognition via Mincle","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTNF is a key proinflammatory cytokine that contributes to various protective immune functions. It can signal via two TNF receptors: TNFR1, which is associated with pro-inflammatory functions, and TNFR2, which is involved in tissue regeneration and cell survival \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Besides its beneficial effects, TNF also plays a crucial role in many chronic inflammatory diseases like rheumatoid arthritis (RA), seronegative spondyloarthropathies, and inflammatory bowel disease (IBD), where a standard treatment strategy involves TNF blockade by antibodies (e.g. Infliximab or Adalimumab) or the human TNFR2-Fc fusion protein Etanercept \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Unwanted side effects of TNF blockade include increased susceptibility to fungal and bacterial infections, including reactivation of latent tuberculosis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In addition, several studies reported an impaired immune response to vaccination due to TNF blockade, including reduced antibody responses to T cell-dependent vaccines for influenza \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and hepatitis B \u003csup\u003e7\u0026ndash;9\u003c/sup\u003e as well as for T cell-independent pneumococcal polysaccharide vaccines \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSubunit vaccines consist of highly purified, often recombinant, antigens that can be processed and presented by antigen-presenting cells (APC) to T cells (signal 1). Their immunogenicity for T cells depends on adjuvants that increase costimulatory molecule expression (signal 2) and production of cytokines to promote and modulate Th cell differentiation (signal 3). Aluminium salts have been used as adjuvants in humans for nearly a century to induce antibody responses, but fail to generate strong T cell responses that are required to protect from intracellular pathogens \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Complete Freund\u0026rsquo;s adjuvant (CFA), an emulsion of heat-killed \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (MTB) in mineral oil, has been used in experimental animals for many decades to induce strong cellular immune responses, including T helper (Th)1 and Th17 cells, but its inflammatory side effects preclude its use in humans \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInducing pathogen-specific Th17, along with Th1 cells, can be beneficial for protection against various intra- and extracellular bacteria and fungi. For example, to clear the extracellular bacterium \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in mice, IL-17 is essential to induce neutrophil recruitment \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. IL-17 activates macrophage killing of \u003cem\u003eBordetella pertussis\u003c/em\u003e and plays a role in the successful vaccination with whole-cell pertussis vaccines \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Besides the recruitment of innate immune cells and the induction of other pro-inflammatory molecules, IL-17 promotes Th1 immunity. For example, it is critical to induce a Th1 response in \u003cem\u003eChlamydia muridarum-\u003c/em\u003einfected mice\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and in humans deficient in Th17 cells (due to a mutation in RORγt, the Th17 lineage-defining transcription factor \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e), an impaired IFNγ response to mycobacteria was observed \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Further, induction of Th17 cells by the only licensed vaccine for tuberculosis, \u003cem\u003eMycobacterium bovis\u003c/em\u003e Bacille Calmette-Gu\u0026eacute;rin (BCG), is required for protection after infection by recruiting Th1 cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, 19\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe progress made in innate immune recognition of infectious danger signals through pattern recognition receptors (PRR) has fertilized the development of next-generation adjuvants that induce and shape both cellular and humoral immune responses \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. One new adjuvant system named CAF01 (Cationic Adjuvant Formulation 01) induces a distinctive, mixed Th1/Th17 response, setting it apart from other next-generation adjuvants \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. CAF01 consists of cationic N,N-dimethyl-N,N-dioctadecylammonium (DDA) liposomes, whose membrane contains the glycolipid trehalose6,6\u0026rsquo;-dibehenate (TDB) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. TDB is a synthetic, less toxic analog of trehalose-6,6\u0026rsquo;-dimycolate (TDM), an abundant and immunostimulatory mycobacterial cell wall glycolipid (\u003cem\u003ealso known as\u003c/em\u003e cord factor). TDM and TDB bind to the activating PRR Mincle (macrophage-inducible C-type lectin) \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The Th17-inducing ability of CAF01 depends on the recognition of its component TDB by Mincle\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and the resulting activation of the Syk-Card9 signaling pathway \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Consistent with the abundance of TDM in the mycobacterial cell wall, the Th17-inducing effect of Complete Freund\u0026rsquo;s adjuvant is strongly reduced in Mincle- and abrogated in Card9-deficient mice \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMincle is expressed on myeloid cells, such as monocytes, macrophages and neutrophils, as well as on some dendritic cells (DC) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Mincle is generally not expressed on adaptive immune cells, but some reports have found it under special circumstances on B cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and T cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In contrast to other PRRs, Mincle is expressed at low levels on resting myeloid cells. In macrophages, stimulation with LPS \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e or the Mincle-ligands TDB and TDM upregulates Mincle expression, leading to enhanced activation and pro-inflammatory signaling \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We previously showed that TDB-/TDM-induced production of TNF was sufficient and essential for Mincle upregulation in macrophages, which was dependent on signaling via TNFR1 \u003csup\u003e32\u003c/sup\u003e. The function of TNF in the induced expression of Mincle is conserved in human monocyte-derived macrophages after stimulation with BCG or LPS \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Upon vaccination in mice, genetic deletion of TNF or pharmacological blockade by Etanercept prevents CAF01-induced Th17 induction, but does not affect the IFNγ response \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile these findings established the essential function of TNF in CAF01-adjuvanted immunization, the cell type on which it acts to enable Th17 differentiation remains undefined. TNF receptors are expressed broadly in different tissues and immune cell types. We hypothesized that TNFR1 may be essential for Th17 induction in our vaccination model because of its pro-inflammatory function and its requirement for Mincle upregulation in macrophages. Conditional TNFR1 knockout mice crossed with cell-type-specific Cre-deleter strains provide a genetically defined system to dissect the pleiotropic effects of TNF \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These mice have been employed to elucidate that for protection against MTB infection, TNFR1 signaling in T cells is dispensable but essential in myeloid cells \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Here, we aimed to determine which immune cell type depends on TNF signaling to elicit both humoral and cellular immune responses following immunization with the recombinant MTB fusion protein H1 \u003csup\u003e36\u003c/sup\u003e adjuvanted with CAF01. Hence, conditional TNFR1 knockout mice were generated using Cre-deleter strains specific for myeloid cells, dendritic cells (DC), or T cells. Furthermore, we employed Mincle-transgenic mice to investigate whether impaired vaccine responses during TNF blockade can be restored by re-establishing robust Mincle expression.\u003c/p\u003e\u003cp\u003eOur results show that TNFR1 on myeloid cells is essential for inducing a Th17 response by H1/CAF01. TNFR1 on DC appears to contribute but is not essential, while TNFR1 on T cells is dispensable. Together, in the H1/CAF01 model, the lack of Mincle upregulation, probably on myeloid cells, can explain most of the effects of the TNF blocker.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTNF blockade selectively prevents the Th17 but not the Th1 response to immunization\u003c/h2\u003e\u003cp\u003eWe previously demonstrated that TNF is required for antigen-specific IL-17 production in mice vaccinated with the TB antigen H1, combined with the Mincle-dependent adjuvant CAF01 \u003csup\u003e32\u003c/sup\u003e. Here, we immunized mice using the same antigen/adjuvant system while blocking TNF with the TNF blocker Etanercept (see Fig.\u0026nbsp;1a), restimulated splenocytes with H1 antigen and analyzed production of IL-17 and IFNγ by CD4\u003csup\u003e+\u003c/sup\u003e T cells using intracellular cytokine staining (Fig.\u0026nbsp;1b). Consistent with our previous ELISA-based results, the frequency of antigen-specific Th17 cells was reduced by TNF blockade during immunization, whereas the H1-specific Th1 response was not affected (Fig.\u0026nbsp;1b; gating strategy shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe next sought to understand whether TNF is necessary to induce or to maintain antigen-specific Th17 cells. To address this question, we utilized Th17 fate reporter mice, in which IL-17A expression results in Cre recombinase-mediated constitutive YFP expression \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Indeed, while only few YFP\u003csup\u003e+\u003c/sup\u003e cells were detected in spleens of naive mice (Fig.\u0026nbsp;1c), CAF01/H1 induced a significant increase of YFP\u003csup\u003e+\u003c/sup\u003e cells, to a percentage comparable to the frequency of IL-17A\u003csup\u003e+\u003c/sup\u003e cells (0.3 vs. 0.5%). In mice immunized under TNF blockade, the number of YFP-marked cells decreased to the level found in unimmunized mice (Fig.\u0026nbsp;1c). In mice immunized without Etanercept, around 30% of YFP-marked cells produced IL-17 after H1 re-stimulation. The YFP-marked cells detected in mice treated with TNF blockade were unable to produce IL-17 after specific restimulation, comparable to unimmunized mice (Fig.\u0026nbsp;1d) Thus, we conclude that TNF is essential for inducing the differentiation of na\u0026iuml;ve T cells into Th17 cells in the H1/CAF01 vaccination model.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePreventing TNF signaling via TNFR1 on myeloid cells reduces the local inflammatory response towards the adjuvant\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTNF affects multiple target cell types and exerts diverse biological effects. It signals through TNFR1 and TNFR2, with TNFR1 being more crucial for its pro-inflammatory function \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. We aimed to determine which immune cell type requires TNFR1 signaling for the induction of Th17 cells. Considering TNFR1's vital role in upregulating the PRR Mincle on innate cells \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, we hypothesized that TNF facilitates the recognition of the adjuvant by antigen-presenting and other innate immune cells. Alternatively, or in addition, an effect on B or T cells could explain the requirement for TNF in the H1/CAF01 vaccination model.\u003c/p\u003e\u003cp\u003eWe utilized cell type-specific deletion of TNFR1 to dissect the mechanistic cellular targets. We initially measured TNFR1 surface expression on immune cells from both immunized and unimmunized wild type (WT) mice (Fig.\u0026nbsp;2b and Fig. S3). We observed no significant TNFR1 expression on B cells. Consequently, we did not further pursue the role of TNFR1 signaling in B cells. TNFR1 was most highly expressed on neutrophils and classical monocytes, while Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes expressed the receptor at a lower level and only on a subset (Fig. S3a). NK cells and NK T cells expressed TNFR1 at a level comparable to Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes. cDC1 (CD88\u003csup\u003e-\u003c/sup\u003e, CD26\u003csup\u003e+\u003c/sup\u003e, CD64\u003csup\u003e-\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003e+\u003c/sup\u003e, XCR1\u003csup\u003e+\u003c/sup\u003e) and cDC2 (CD88\u003csup\u003e-\u003c/sup\u003e, CD26\u003csup\u003e+\u003c/sup\u003e, CD64\u003csup\u003e-\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003e+\u003c/sup\u003e, XCR1\u003csup\u003e-\u003c/sup\u003e) as well as T cells (CD4 T cells, CD8 T cells and γδ T cells) showed a lower expression level. On macrophages (Ly6G\u003csup\u003e-\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003elow\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003e) only a small subpopulation showed a detectable TNFR1 expression.\u003c/p\u003e\u003cp\u003eFirst, we checked the importance of TNF signaling via TNFR1 on myeloid cells. To this end, we crossed LysM-Cre mice\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e with mice in which exons 2\u0026ndash;5 of the TNFR1 are floxed (Tnfrsf1a\u003csup\u003etm3.3Gkl\u003c/sup\u003e, \u003csup\u003e34\u003c/sup\u003e). This LysM-Cre-mediated deletion of the TNFR1 resulted in the complete loss of TNFR1 expression on neutrophils and a substantial reduction on Ly6C\u003csup\u003ehigh\u003c/sup\u003e inflammatory monocytes (Fig.\u0026nbsp;2b). On Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes and on macrophages, TNFR1 was not reduced significantly by LysM-Cre mediated deletion. TNFR1 was expressed on subpopulations of Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes and F4/80\u003csup\u003e+\u003c/sup\u003e macrophages (Fig. S3a). Although LysM-Cre completely abolished TNFR1 staining in these subpopulations, MFI values for Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes were not affected (Fig.\u0026nbsp;2b). In all other tested cell types, TNFR1 expression was not significantly changed, except for a slight increase on NK cells and a reduction on NK T cells. We will address the potential importance of TNFR1 on NK and NK T cells later.\u003c/p\u003e\u003cp\u003e We first studied the effect of TNFR1 deficiency in myeloid cells on the local inflammatory response to immunization with H1/CAF01 (for the experimental layout see Fig.\u0026nbsp;2a). CAF01, a liposome-based adjuvant, forms a depot at the site of injection. Innate immune cells migrate to this location and slowly transport the adjuvant and antigen to the draining lymph node \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Upon s.c. immunization in the footpad, the measurement of its thickness over time can serve as a proxy for immune cell infiltration and inflammation at the site of injection. The robust footpad swelling after H1/CAF01 injection was reduced by approximately 50% in myeloid TNFR1 knockout (KO) mice compared to the control group (Fig.\u0026nbsp;2c), which corresponds well to the reduction observed in \u003cem\u003eTnf\u003c/em\u003e\u003csup\u003e-/-\u003c/sup\u003e mice \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, indicating that TNF signaling in myeloid cells plays a significant role in the local inflammatory response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTNFR1 signaling in myeloid cells is essential for the antigen-specific Th17 response\u003c/h3\u003e\n\u003cp\u003eThe absence of TNFR1 in myeloid cells completely prevented the induction of H1-specific production of IL-17 by LN cells, while the production of IFNγ and IL-10 remained unaltered (Fig.\u0026nbsp;2d, similar results observed for spleen cells see Fig. S4a). Of note, neither the floxing of TNFR1 alone (Fig. S5a) nor the insertion of the Cre recombinase under the LysM promoter (Fig. S5b) influenced the IL-17 production after immunization.\u003c/p\u003e\u003cp\u003eThe reduced IL-17 production was accompanied by the vaccine's inability to induce H1-specific Th17 cells in the myeloid TNFR1 KO mice, whereas the induction of Th1 cells was unaffected (Data from LN in Fig.\u0026nbsp;2e, comparable results were observed for spleen Fig. S4b, c).\u003c/p\u003e\u003cp\u003eIn summary, the absence of TNFR1 on myeloid cells abrogates the Th17 response induced by the Mincle-activating adjuvant CAF01, replicating the effect of genetic whole body deletion of the \u003cem\u003eTnf\u003c/em\u003e gene as we showed before \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eTNF contributes to antibody induction, but TNFR1 on myeloid cells is dispensable\u003c/h3\u003e\n\u003cp\u003eWe also tested the consequence of genetic deletion or pharmacologic blockade of TNF versus the specific knockout of TNFR1 in myeloid cells for the induction of the humoral immune response. To do so, we measured H1-specific antibody (Ab) titers after two injections of H1/CAF01 (Experimental layout Fig. S2a). Knockout of TNF reduced titers of all studied isotypes by more than one order of magnitude (Fig. S2b). It is well established that the formation of germinal centers is impaired in \u003cem\u003eTnf\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Transient pharmacological TNF blockade of the response to vaccination by sequential treatment with Etanercept and a neutralizing anti-TNF antibody caused a weaker but significant reduction in H1-specific antibody (Ab) titers of the proinflammatory isotypes IgG2c and IgG2b, but not of IgG1 (Fig. S2b). In contrast to the effect of genetic deletion or pharmacological inhibition of TNF, the myeloid cell-specific TNFR1 KO did not affect the induction of H1-specific Ab after immunization (Fig. S2c).\u003c/p\u003e\u003cp\u003e\u003cem\u003eTNFR1 on DC plays a limited role in the antigen-specific Th17 response and is dispensable for induction of H1-specific antibodies\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAfter we found that the TNFR1 on myeloid cells is essential for the Th17 response, we wanted to test the importance of the TNFR1 on DCs. For this, we employed two well-characterized DC-targeting Cre-deleter mouse lines. First, a Cre knock-in at the Clec9a locus specifically deletes \u003cem\u003eloxP\u003c/em\u003e-flanked genes in DC progenitors \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In Clec9a-Cre\u003csup\u003ehet\u003c/sup\u003e; TNFR1\u003csup\u003efl/fl\u003c/sup\u003e mice, we observed a complete absence of TNFR1 staining on cDC1 and a significant, but incomplete reduction (ca. 65%) of TNFR1 surface protein on cDC2 (Fig.\u0026nbsp;3a, representative histograms Fig. S6a). Besides DCs, there was a reduction of TNFR1 expression on macrophages and on NK cells, whilst the remaining tested cell types had intact TNFR1 expression. We will address the importance of NK cells on the induction of an antigen-specific Th17 response later.\u003c/p\u003e\u003cp\u003eTo rule out the possibility that the remaining TNFR1 expression on cDC2 leads to an incorrect conclusion, we used CD11c-Cre-mediated deletion\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e of TNFR1 in addition. In this model, TNFR1 staining was completely absent on cDC1 and cDC2 (Fig.\u0026nbsp;3b, representative histograms Fig. S6b). In contrast to the Clec9a-Cre model, there was also a reduction in TNFR1 surface protein across all T cell subsets. Additionally, a significant reduction of TNFR1 was observed in Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes and in macrophages.\u003c/p\u003e\u003cp\u003eIn both models for deleting TNFR1 on DC, the inflammatory response at the site of injection, measured by the footpad swelling, was unaffected (Fig.\u0026nbsp;3c, d).\u003c/p\u003e\u003cp\u003eClec9a-mediated TNFR1 deletion did not alter cytokine production after the restimulation of LN cells from immunized mice (Fig.\u0026nbsp;3e). These findings were confirmed by the unaltered induction of Ag-specific Th17 and Th1 cells after immunization (Fig.\u0026nbsp;3f). This suggests that TNFR1 expression on cDC1 cells is not required for H1/CAF01 induced Th17 responses.\u003c/p\u003e\u003cp\u003eThe CD11c-Cre-mediated complete TNFR1 deletion on all DC subsets did not affect the production of IFNγ and IL-10 in LN cells (Fig.\u0026nbsp;3g) nor the induction of Th1 cells (Fig.\u0026nbsp;3h). In contrast, it led to a significant reduction in IL-17 production upon restimulation of LN cells with H1, with 75\u0026ndash;80% reduction when measured by ELISA (Fig.\u0026nbsp;3g), or around 50% reduction in LN CD4\u0026thinsp;+\u0026thinsp;T cells producing IL-17A, as measured by intracellular cytokine staining (Fig.\u0026nbsp;3h).The same pattern was observed after restimulation of spleen cells and measurement by ELISA (Fig. S8a, c) and after intracellular cytokine staining of spleen cells (Fig. S8b, d). No significant inhibition of IL-17 production was observed in CD11c-Cre\u003csup\u003e+\u003c/sup\u003e; TNFR1\u003csup\u003ewt/wt\u003c/sup\u003e mice (Fig. S5c). In summary, TNFR1 on DC seems to play a role in inducing a Th17 response after H1/CAF01 immunization, but the Th17 response does not entirely depend on it, as observed for the TNFR1 on myeloid cells.\u003c/p\u003e\u003cp\u003eWe also measured the H1-specific Ab titer in H1/CAF01-immunized CLec9a-Cre\u003csup\u003ehet\u003c/sup\u003e; TNFR1\u003csup\u003efl/fl\u003c/sup\u003e and CD11c-Cre\u003csup\u003e+\u003c/sup\u003e; TNFR1\u003csup\u003efl/fl\u003c/sup\u003e mice. In both models, the specific Ab titers were similar to those in the control group (Fig. S2c). This implies that TNFR1 on DC is dispensable for the H1/CAF01-induced Ab response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eTNFR1 on T cells is not required for the antigen-specific Th17 response\u003c/h3\u003e\n\u003cp\u003eThe impaired Th17 response in CD11c-Cre\u003csup\u003e+\u003c/sup\u003e; TNFR1\u003csup\u003efl/fl\u003c/sup\u003e mice was associated with a reduction in TNFR1 surface protein on T cells (Fig.\u0026nbsp;3b), raising the question whether TNFR1 on T cells is required for Th17 differentiation in our immunization model. To address this, we used the Lck-Cre mouse \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e to delete the floxed TNFR1 during thymic development of T cells.\u003c/p\u003e\u003cp\u003eIn this model, TNFR1 was no longer present on the surface of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells as well as on γδ T cells and NK T cells (Fig.\u0026nbsp;4a, representative example see Fig. S7). Besides this, TNFR1 expression on all other studied cell types was unaffected. Thus, Lck-Cre-mediated deletion was efficient and specific in the T cell compartment.\u003c/p\u003e\u003cp\u003eOf note, a lower frequency of CD4\u003csup\u003e+\u003c/sup\u003e T cells was observed in na\u0026iuml;ve and immunized Lck-Cre\u003csup\u003e+\u003c/sup\u003e; TNFR1\u003csup\u003efl/fl\u003c/sup\u003e mice (Fig.\u0026nbsp;4b). The local inflammatory response in the footpad remained unchanged (Fig.\u0026nbsp;4c). The absence of TNFR1 did not significantly affect IL-17 production following the restimulation of LN cells from immunized mice (Fig.\u0026nbsp;4d, comparable to spleen cells Fig. S9a). However, a significant reduction in IFNγ and IL-10 production was noted. The frequency of induced specific Th17 and Th1 cells in the LN remained unchanged (Fig.\u0026nbsp;4e, spleen comparable see Fig. S9b).\u003c/p\u003e\u003cp\u003eIn conclusion, the TNFR1 on T cells may contribute to their overall numbers or function, but is not essential for the induction of specific Th17 cells by the H1/CAF01 vaccine.\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe adjuvant CAF01 induced Th17-polarizing cytokines in monocytes in a TNF-dependent manner.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe previously found that CCR2\u003csup\u003e+\u003c/sup\u003e monocytes, but not neutrophils, are critical for IL-17 induction by CAF01 \u003csup\u003e44\u003c/sup\u003e. Therefore, we focused on monocytes isolated from mouse bone marrow cells. We observed, that there was a TNF-dependent transcriptional upregulation of Mincle after stimulation with the Mincle ligand TDB (contained in CAF01) as well as with the mycobacterial cord factor TDM (Fig.\u0026nbsp;5a). The expression of IL-6 and IL-1β was TNF-dependently upregulated by both Mincle ligands (Fig.\u0026nbsp;5b, c, d).\u003c/p\u003e\u003cp\u003eIn summary, we show that the CAF01 constituent TDB induced the Th17 polarizing cytokines IL-6 and Il-1β in monocytes, probably due to the TNF-dependent upregulation of Mincle.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTransgenic constitutive expression of Mincle uncouples its surface expression from TNF signaling\u003c/p\u003e\u003cp\u003eNext, we aimed to assess whether the impaired Mincle upregulation in mice treated with Etanercept or lacking TNFR1 in myeloid cells is causal in the abrogation of Th17 induction after vaccination. To achieve this, we used a transgenic mouse model in which Mincle is constitutively expressed at a high level (Mincle\u003csup\u003etg\u003c/sup\u003e, \u003csup\u003e45\u003c/sup\u003e). Additionally, to rule out confounding factors due to the regulation of endogenous Mincle by TNF signaling, the Mincle\u003csup\u003etg\u003c/sup\u003e mice were crossed onto a Mincle-deficient background (\u003cem\u003eClec4e\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/\u003c/em\u003e-\u003c/sup\u003e, further referred to as Mincle\u003csup\u003e-/- 46\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eTo determine Mincle regulation in these mice, bone marrow-derived macrophages (BMDM) were generated and stimulated with the Mincle ligand TDM (Fig.\u0026nbsp;6a, representative Histogramms Fig. S10a). Mincle was expressed at low levels on resting WT BMDM and as expected strongly upregulated in a TNF-dependent manner after stimulation. In Mincle\u003csup\u003e-/-\u003c/sup\u003e; Mincle\u003csup\u003etg\u003c/sup\u003e BMDM, Mincle was highly expressed under resting conditions, and its expression level was unaffected by the TNF blocker Etanercept.\u003c/p\u003e\n\u003ch3\u003eTNF-independent, high constitutive Mincle expression can rescue Th17 induction\u003c/h3\u003e\n\u003cp\u003eWe immunized the Mincle\u003csup\u003e-/-\u003c/sup\u003e; Mincle\u003csup\u003etg\u003c/sup\u003e mice to test the effect of TNF block when Mincle expression is no longer regulated (Experimental design Fig.\u0026nbsp;6b). The local inflammatory response, measured by footpad swelling, was reduced in WT mice due to TNF block (Fig.\u0026nbsp;6c). The magnitude of this reduction was comparable to that seen in mice lacking TNFR1 on myeloid cells. In the transgenic mice, footpad swelling was greater compared to WT mice. TNF blockade with Etanercept reduced it, but only to the level seen in non-TNF-blocked WT mice.\u003c/p\u003e\u003cp\u003eIL-17 production after restimulation of LN cells from immunized mice was, as expected, entirely prevented in TNF-blocked WT mice (Fig.\u0026nbsp;6d, comparable results for spleen cells, see Fig. S10b). In contrast, IL-17 production was restored in TNF-blocked transgenic mice to the level observed in immunized WT mice. The production of IFNγ and IL-10 was not altered due to the TNF block. Correspondingly, the frequency of specific Th17 cells was reduced in immunized TNF-blocked WT mice, whereas in Mincle-transgenic mice, the TNF blocker did not affect the frequency of Th17 cells compared to immunized WT mice (Fig.\u0026nbsp;6e, comparable results for spleen cells, see Fig. S10c, d).\u003c/p\u003e\u003cp\u003eUnder physiological conditions, Mincle is expressed on myeloid cells and some dendritic cells (DC) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Some reports have found it under special circumstances on B cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and T cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We now tested the expression of the transgenic mincle on celltypes which are not known to express mincle in WT mice. We found that, NK and NK T cells from Mincle\u003csup\u003e-/-\u003c/sup\u003e; Mincle\u003csup\u003etg\u003c/sup\u003e mice expressed significant Mincle protein on the surface, in contrast to their WT counterparts (Fig. S11a). This observation raised the question whether NK and NK T cells contributed to the observed rescue of Th17 induction. Therefore, we tested whether NK1.1-positive cells contribute critically during immunization in WT and transgenic mice under TNF blockade by repeatedly injecting the anti-NK1.1 antibody PK136 (see experimental scheme in Fig. S11b), resulting in the complete depletion of NK1.1-positive cells (Fig. S11c).\u003c/p\u003e\u003cp\u003eThe immunization of WT mice in the absence of NK1.1-positive cells still led to IL-17 induction (Fig. S11d), with levels comparable to those observed in previous experiments. Also, the complete prevention of specific IL-17 production due to TNF block during immunization did not depend on NK1.1-positive cells. Therefore, we can exclude a potential impact of the observed differences in TNFR1 expression on NK and NK T cells after Clec9a-Cre-mediated deletion (Fig.\u0026nbsp;3a) or CD11c-Cre-mediated deletion (Fig.\u0026nbsp;3b). In addition, the rescue of the IL-17 production in TNF-blocked Mincle\u003csup\u003e-/-\u003c/sup\u003e; Mincle\u003csup\u003etg\u003c/sup\u003e mice was unaffected by the absence of NK1.1-positive cells (Fig. S11d). Together, the Th17 rescue effect observed in the transgenic mice most likely originates from the constitutive expression of Mincle on either monocytes or DC, since NK1.1-positive cells were dispensable and transgenic Mincle was not stably expressed on T and B cells (Fig. S11a).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe employed conditional TNFR1-deficient mice to dissect the mechanism underlying our previous observation that TNF is required for Th17 induction by the Mincle-dependent adjuvant CAF01 \u003csup\u003e32\u003c/sup\u003e. The complete loss of IL-17-producing Th cells in mice lacking TNFR1 in myeloid cells reproduced the effect of whole-body TNF deficiency and confirmed the selective loss of Th17, but not Th1, differentiation. Although TNFR1 is highly expressed in monocytes and in neutrophils, our previous demonstration that CAF01-induced Th1/Th17 induction requires CCR2\u003csup\u003e+\u003c/sup\u003e monocytes, but not Ly6G\u003csup\u003e+\u003c/sup\u003e neutrophils \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, allows us to conclude that only TNFR1 on monocytes or macrophages is essential for Th17 induction. TNFR1 deletion in DC by CD11c-Cre resulted in a significant but incomplete reduction in IL-17-producing CD4\u003csup\u003e+\u003c/sup\u003e T cells, whereas Clec9a-Cre-mediated deletion had no effect on Th cell differentiation at all. Finally, deletion of TNFR1 in T cells did not impair Th17 induction at all. Together, the use of cell type-specific TNFR1 knockout mouse lines clearly revealed that monocytes/macrophages are the critical target cells for promoting Th17 induction by TNF. Mechanistically, the partial restoration of Th17 adjuvanticity in Mincle\u003csup\u003e−/−\u003c/sup\u003e; Mincle\u003csup\u003etg\u003c/sup\u003e mice undergoing TNF blockade indicates that downregulation of Mincle expression contributes substantially to the loss of Th17 differentiation by impairing myeloid cell sensing of the adjuvant TDB.\u003c/p\u003e\u003cp\u003eIn contrast to the Th17 response, and consistent with our previous finding that IFNγ responses were unaffected by TNF \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, we found that Th1 differentiation is independent of TNF. In this context, it is of interest that DDA liposomes, in which the Mincle ligand TDB would be incorporated to obtain CAF01, possess an intrinsic, moderate IFNγ- but not IL-17-inducing adjuvant activity, which is independent of Mincle \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, the Mincle-dependent adjuvant activity of CAF01 is more crucial for inducing the Th17 response but not the Th1 response.\u003c/p\u003e\u003cp\u003eActivation and priming of naïve T cells require MHC-restricted antigen recognition by the TCR (signal 1) in conjunction with costimulation (signal 2). TNF and TNFR1 signaling appear not to be required for T cell priming \u003cem\u003ein vivo\u003c/em\u003e by the recombinant H1/CAF01 vaccination, as the induction of antigen-specific Th1 cells and the secretion of IFNγ and IL-10 upon restimulation \u003cem\u003ein vitro\u003c/em\u003e were not affected by genetic deficiency or pharmacological blockade. However, the differentiation of Th cells is controlled by the cytokine environment in the draining lymph node and the spleen (signal 3). IL-1β, IL-6, IL-23 and TGFβ promote Th17 differentiation, while type 1 interferons, IFNγ, and IL-4 inhibit it \u003csup\u003e47\u003c/sup\u003e. We previously showed that IL-1 receptor signaling via Myd88 is required for the Th17-inducing effect of CAF01 \u003cem\u003ein vivo\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Here, we found that the TNF blocker Etanercept strongly impaired the expression of IL-6 and IL-1β in monocytes stimulated with the Mincle ligand TDB \u003cem\u003ein vitro\u003c/em\u003e. This reduction in Th17-inducing cytokines complements the \u003cem\u003ein vivo\u003c/em\u003e results showing that TNFR1-deficiency in myeloid cells abrogates Th17 induction, and supports the interpretation that TNFR1 signaling drives the activation of monocytes and macrophages that generate signal 3. In contrast, TNFR1 signaling in DC seems less important for Th17 differentiation, with intact Th17 responses after complete TNFR1 deletion on cDC1s, and a reduced capacity to produce IL-17 following CD11c-Cre-mediated complete deletion of the TNFR1 on all DC subsets. Together, the results from using LysM-Cre, Clec9a-Cre and CD11c-Cre deleter mice suggest that antigen presentation by cDC is not regulated by TNF, whereas signal 3 is derived mainly from monocytes/macrophages, and perhaps cDC2, activated by the Mincle-dependent adjuvant and depends on TNFR1 signaling.\u003c/p\u003e\u003cp\u003eWhile the specificity of Cre-mediated TNFR1 deletion in different immune cells was overall according to our expectations, the distinction between monocytes, macrophages and different DC subsets using the existing markers is not absolute and subject to adjustments as new insights are published. In relation to our results, the description of the DC3 lineage by Ginhoux and colleagues \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e as Th17-inducing cells derived from Ly6C\u003csup\u003e+\u003c/sup\u003e monocyte-DC precursors is of special interest because fate mapping in LysM-Cre-R26TdT mice showed a high proportion of marked DC3. It is therefore possible that the myeloid cell populations required for Th17 induction, which we considered as monocytes/macrophages, may also contain DC3. It was reported before, that CD11c-Cre shows leaky expression in different hematopoietic lineages, that can lead to deletion of sensitive floxed alleles \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe observed a partial deletion of TNFR1 on macrophages and Ly6Clow monocytes by CD11c-Cre, therefore it is also possible that the observed reduction in Th17 cells is not caused by the deletion of TNFR1 in cDCs, and that the cytokines secreted by TNFR1-activated macrophages and Ly6Clow monocytes are critical for Th17 differentiation.\u003c/p\u003e\u003cp\u003eBased on reports showing that TNF block can reduce Th17 and Th1 cell differentiation after anti-CD3/CD28-induced differentiation of naïve human T cells \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, we considered that TNF may act directly on CD4\u003csup\u003e+\u003c/sup\u003e T cells to promote Th17 differentiation and therefore included the Lck-Cre deleter strain. TNFR1 surface protein levels were relatively modest in T cells and we found no evidence for the importance of the TNFR1 on T cells in Th17 induction. However, we note that the effects of TNF on T cells can depend on signaling through TNFR2, which we did not address here. First, it was described in human T cells that TNF signaling via TNFR2 but not TNFR1 acts as a costimulatory signal for T cell receptor activation \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In addition, a recent publication revealed that TNF signaling through TNFR2 together with TGFβ enhances Th17 induction \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In the same paper, it was demonstrated that IL-1β induces TNF in T cells, and that, in contrast to IL-6, TNFR2 is essential for the IL-1β effect on Th17 differentiation. Clearly, the potential involvement of TNFR2 in regulation of vaccination-induced Th cell differentiation deserves further investigation.\u003c/p\u003e\u003cp\u003eBesides the direct effects on T cells, it was shown in rheumatoid arthritis (RA) patients that TNF induces IL-6 in monocytes, via TNFR1 and TNFR2, which leads to an increase in Th17 cells \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. This reflects our finding that Etanercept prevents the induction of IL-6 in murine monocytes. Another paper described that TNF promotes differentiation of human monocytes into DC, which in turn have the capacity to induce Th17 responses \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Alternatively, an indirect effect was described in RA patients treated with the TNF blocker Adalimumab (but not observed for Etanercept) that regulatory T cells increase, which inhibit IL-6 production in monocytes via IL-10 \u003csup\u003e57\u003c/sup\u003e. We did not find increased IL-10 levels in mice after TNF blockage by Etanercept, arguing that this mechanism may be specific to RA or humans, or that the type of TNF blocker makes a difference.\u003c/p\u003e\u003cp\u003eIn addition to the Th cell response, we analyzed whether TNF/TNFR1 signaling contributes to the generation of specific antibodies after H1/CAF01 immunization. It is known that the knockout of TNF in mice prevents germinal center (GC) formation, disrupts follicular DC networks and consequently leads to impaired antibody responses \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Even transient blockade of TNF by Etanercept can have an adverse effect on GC organization and reduce the number of follicular DC \u003csup\u003e59\u003c/sup\u003e. TNFR1 contributes to GC formation through its role in FDC differentiation \u003csup\u003e606162\u003c/sup\u003e. In the H1/CAF01 immunization model, we found that induction of H1-specific antibodies was markedly reduced in whole body TNF knockout mice and blockade of TNF during the immunization period was moderately inhibitory. In contrast, TNFR1 deletion in myeloid cells and in cDC did not affect antibody titers. Thus, the loss of antigen-specific Th17 cells observed in the myeloid TNFR1 knockout mice was not linked to antibody responses, indicating that, although Th17 cells can act \u003cem\u003ein vitro\u003c/em\u003e as B cell helpers and drive an isotype switch towards IgG2a and IgG3 \u003csup\u003e63\u003c/sup\u003e, the IgG2a response to H1/CAF01 is likely driven by Th1 cells \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe previously linked the requirement of TNF for the induction of an IL-17 response after immunization with the Mincle-dependent adjuvant CAF01 to upregulation of this PRR \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We have now found that TNFR1 signaling on myeloid cells is essential for Th17 induction but dispensable for Th1 or humoral responses. We therefore postulate that the missing upregulation of Mincle after binding the adjuvant interrupts a positive TNF-dependent feed-forward loop, thwarting myeloid cell activation and secretion of Th17-polarizing cytokines. It was found that in Mincle KO mice, the induction of IL-6, IL-1β, and IL-23 by CFA was reduced, concomitant with a strongly reduced Th17 response \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In line with our model, we found that after stimulation of monocytes with the CAF01 component TDB, the increase in Mincle expression was prevented by blocking TNF, which was accompanied by the prevention of the TDB-induced expression of IL-6 and IL-1β. Significantly, constitutive transgenic Mincle expression prevented the loss of the specific Th17 response caused by the TNF blocker, further supporting the notion that disrupted upregulation of Mincle in myeloid cells impairs the sensing of the TDB adjuvant.\u003c/p\u003e\u003cp\u003eThe Mincle-transgenic mouse line was used before to study the role of Mincle signaling in pneumonia caused by pneumococci and by \u003cem\u003eStaphylococcus aureus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. A limitation of this mouse model is the ubiquitous expression of transgenic Mincle mRNA that contrasts the specific expression of endogenous Mincle in myeloid cells. However, surface localization of Mincle protein requires the presence of the adapter protein Fc receptor gamma chain (encoded by \u003cem\u003eFcer1g\u003c/em\u003e) that interacts with a conserved arginine residue in the transmembrane domain of Mincle \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Fc receptor gamma is expressed in a more restricted pattern on myeloid cells, mast cells, and innate lymphoid cells, including NK and NKT cells. Indeed, in addition to myeloid cells and some DC, we found robust transgenic Mincle protein expression on NK1.1\u003csup\u003e+\u003c/sup\u003e cells, but these were not contributing to Th17 induction as shown by antibody-mediated depletion experiments.\u003c/p\u003e\u003cp\u003eIn summary, we found that Mincle upregulation is a mechanism through which TNF enhances adjuvant recognition and responsiveness. This process is crucial in myeloid cells, especially in monocytes, where the lack of TNFR1 signaling prevents the secretion of Th17-promoting cytokines. Besides the Mincle-dependent adjuvant CAF01, the effect of TNF on Mincle regulation on myeloid cells could also be relevant for other, clinically used vaccines. The widely used aluminum salt-based adjuvants stimulate the immune system by inducing cell death \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Mincle binds molecules released by dying cells, such as spliceosome-associated protein 130 (SAP130)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e and β-glucosylceramide \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Therefore, Mincle and its regulation by TNF may be essential for recognizing these aluminium adjuvant-induced DAMPs. In addition, the pneumococcal antigen glycolipid glucosyl-diacylglycerol (Glc-DAG) was identified to bind to Mincle \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Whether Glc-DAG is present in pneumococcal polysaccharide vaccine preparations and acts as a built-in Mincle-dependent adjuvant remains to be determined. If so, the reduced immunogenicity of the pneumococcal polysaccharide vaccine\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e in patients receiving TNF blockers could be caused by a similar mechanism. Therefore, further studies on the role of Mincle in the adverse effects of TNF blockade on response to different vaccine adjuvants are necessary. This knowledge will be valuable in gaining a fundamental understanding of the mechanisms of action, which are essential for the further design and application of vaccines and adjuvants.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMice\u003c/p\u003e\u003cp\u003eAll mice were bred and housed under specific pathogen-free conditions at the Präklinische Experimentelle Tierzentrum of the Medical Faculty in Erlangen, Germany, or at the Statens Serum Institute, Denmark. Frozen sperm cells of TNFR1\u003csup\u003efl/wt\u003c/sup\u003e mice\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e were obtained from the European Mouse Mutant Archive in Athens, Greece (EMMA strain 11019, B6.129P2(Cg)-Tnfrsf1a\u003csup\u003etm3.3Gkl/Flmg\u003c/sup\u003e, archived on a C57BL/6 background), and used to re-derive the mice by \u003cem\u003ein vitro\u003c/em\u003e fertilization at the Transgenic Facility of the Friedrich-Alexander-University Erlangen-Nürnberg. To generate cell type-specific conditional TNFR1 KO lines, TNFR1\u003csup\u003efl/fl\u003c/sup\u003e mice were crossed with LysM-Cre (B6.Lyz2\u003csup\u003etm1(cre)Ifo\u003c/sup\u003e; \u003csup\u003e38\u003c/sup\u003e), Clec9a-Cre B6J.B6N(Cg)-Clec9atm2.1(icre)Crs/J; \u003csup\u003e41\u003c/sup\u003e), CD11c-Cre (B6.Cg-Tg(Itgax-cre)1-1Reiz/J; \u003csup\u003e42\u003c/sup\u003e), or Lck-Cre (Tg(Lck-cre)1Jtak \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e) mice. Mincle\u003csup\u003etg\u003c/sup\u003e mice \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e were obtained from Sho Yamasaki and crossed with Mincle KO mice (Clec4e\u003csup\u003etm1.1Cfg\u003c/sup\u003e) generated and provided by the Consortium for Functional Glycomics \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eTnf\u003c/em\u003e\u003csup\u003e\u003cem\u003e−/−\u003c/em\u003e\u003c/sup\u003e mice\u003csup\u003e40\u003c/sup\u003e were provided by Dr. Ulrike Schleicher. C57BL/6N mice were purchased from Charles River Laboratories. At the end of the experiment the mice were euthanized by cervical dislocation. All mouse experiments were approved by the \u003cem\u003eRegierung von Unterfranken\u003c/em\u003e (protocol number 55.2.2-2532-2-1641).\u003c/p\u003e\u003cp\u003eIL-17A fate reporter mice were made by crossing the Il17a\u003csup\u003etm1.1(icre)Stck\u003c/sup\u003e/J (IL-17cre) strain with the B6.129X1-Gt(ROSA)26Sor\u003csup\u003etm1(EYFP)Cos\u003c/sup\u003e/J (R26R-EYFP) strain \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e (from The Jackson Laboratory (Bar Harbor, USA)) and handled at the experimental animal facility at Statens Serum Institut. At the end of the experiment the mice were euthanize by cervical dislocation. Experimental work was conducted in accordance with the regulations of the Danish Ministry of Justice and the Danish National Experiment Inspectorate under permit 2017-15-0201-01363 and in compliance with the European Community Directive 2010/63 EU for the care and use of laboratory animals.\u003c/p\u003e\u003cp\u003eImmunizations\u003c/p\u003e\u003cp\u003eTo study the cellular immune response, we used a 7-day immunization protocol, as previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Mice were immunized s.c. in both footpads with 50 µl CAF01 \u003csup\u003e21\u003c/sup\u003e mixed with 1 µg H1 \u003csup\u003e36\u003c/sup\u003e per foot, except in the IL-17A fate-reporter mice, where s.c. immunization was performed at the base of tail using a 100µl volume. Unimmunized mice were injected with the same volume of PBS. All footpad immunizations were performed on unconscious animals after inhalation anesthesia with 4% Isoflurane. For the immunization in the base of tail (s.c.) no anesthesia was used. After footpad immunization, the footpad thickness was measured before the immunization (day 0) and every second day after immunization. The footpad swelling was calculated by subtracting the initial thickness (on day 0) from the later obtained values. On day 7 after immunization, the mice were killed, and the inguinal and popliteal lymph nodes as well as the spleen were isolated.\u003c/p\u003e\u003cp\u003eFor the experiments requiring TNF blockade, Etanercept (3 mg/kg in 100 µl PBS) was injected s.c. in the flank (without anesthetization) on the day of immunization and every other day thereafter. The control group was injected in parallel with 100 µl PBS.\u003c/p\u003e\u003cp\u003eFor the depletion of NK1.1-positive cells, an anti-NK1.1 antibody (clone PK136, Leinco Technologies) was used in addition to etanercept treatment in the 7-day immunization protocol. 250 µg/mouse of this antibody or an isotype control (clone C1.18.4, Leinco Technologies) was injected i.p. (without anesthetization) one day before and a second time two days after immunization with H1/CAF01.\u003c/p\u003e\u003cp\u003eTo study the humoral immune response, a 5-week immunization protocol was used. Here, the mice received a second immunization at day 21 (2 µg H1 in 100 µl CAF01, s.c. base of tail). Blood was collected at day 0 (no H1-specific Ab detectable, data not displayed) and after the killing on day 36.\u003c/p\u003e\u003cp\u003eTo assess the role of TNF on the Ab production, additionally etanercept (3 mg/kg in 100 µl PBS) was injected s.c. in the flank on the day of immunization and every other day until day 18. Then, to prevent hindrance of the TNF block by the formation of anti-etanercept Ab, we switched to injecting anti-murine TNF Ab (200 µg/mouse, clone TN3-19.12, Leinco Technologies) every second day starting from day 20. A control group was injected with a matching isotype control (clone PIP, Leinco Technologies). All s.c. and i.p. injections of antibodies and etanercept were performed without anesthetization. In this protocol, the mice were killed 1 week after the booster immunization and blood was collected.\u003c/p\u003e\u003cp\u003eDifferentiation and stimulation of BMDM\u003c/p\u003e\u003cp\u003eBone marrow cells were isolated from C57BL/6 mice and Mincle\u003csup\u003e−/−\u003c/sup\u003e; Mincle\u003csup\u003etg\u003c/sup\u003e mice and differentiated as described before \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. TDM was dissolved in isopropanol and coated to the bottom of the well (final concentration of 2 µg/ml) by allowing the isopropanol to evaporate. For the control wells, the same volume of isopropanol was evaporated. The BMDM were seeded in a flat-bottom 96-well plate (2*10\u003csup\u003e5\u003c/sup\u003e cells/well). Etanercept 100 µg/ml was added to the respective wells. The cells were stimulated for 24 h.\u003c/p\u003e\u003cp\u003eIsolation and stimulation of BM monocytes\u003c/p\u003e\u003cp\u003eTo isolate monocytes from the bone marrow, the monocyte Isolation Kit (Miltenyi Biotec, Order No.: 130-100-629) was used according to the instructions of the producer. BM monocytes were stimulated by seeding them into a flat-bottom 96-well plate (2*10\u003csup\u003e5\u003c/sup\u003e cells/well). The wells were coded before with isopropanol, TDB (5 µg/ml), or TDM (2 µg/ml) as described above. Etanercept (final concentration 100 µg/ml) was added to the respective wells. The cells were stimulated for 24 h.\u003c/p\u003e\u003cp\u003eRestimulation of cells from spleen and lymph node\u003c/p\u003e\u003cp\u003eA single cell suspension of lymph node cells was obtained by mechanically dissociating the LN using a 70 µm cell strainer. The spleen was also mashed through a 70 µm strainer, and additionally, the erythrocytes were lysed using ammonium chloride. 5 x 10\u003csup\u003e5\u003c/sup\u003e LN and spleen cells were restimulated in a 96-well U-bottom cell culture plate for 96h with H1 (1 µg/ml).\u003c/p\u003e\u003cp\u003eCytokine ELISA\u003c/p\u003e\u003cp\u003eCytokines were measured in the supernatants of restimulated cells. For this purpose, the following ELISA kits were used according to the manufacturer's instructions: IFNγ (R\u0026amp;D Systems, DY485), IL-17 (R\u0026amp;D Systems, DY421), IL-10 (R\u0026amp;D Systems, DY417), and IL-6 (R\u0026amp;D Systems, DY406).\u003c/p\u003e\u003cp\u003eFlow cytometry\u003c/p\u003e\u003cp\u003eAfter blocking with anti-mouse CD16/32 antibody (eBioscience) the dead cells were stained with the Fixable Viability Dye eFluor 506 (Invitrogen 65-0866-14). Next, fluorophore-coupled antibodies were used to stain for the surface markers (see Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e for a list of the used antibodies). For the staining of Mincle, an uncoupled primary antibody (MBL, clone 4A9) was used and detected by using a suitable fluorescent-labeled secondary antibody.\u003c/p\u003e\u003cp\u003eFor intracellular cytokine staining, 1.5 x 10\u003csup\u003e6\u003c/sup\u003e cells were restimulated in a 96-well plate using 5 µg/ml of each of the three H1 peptides Ag85B \u003csub\u003ep241−255\u003c/sub\u003e, Ag85B \u003csub\u003ep261−280\u003c/sub\u003e, ESAT-6 \u003csub\u003ep1−15\u003c/sub\u003e (obtained from Peptides and Elephants) or using the whole H1 protein. The cells were stimulated for one hour, followed by five hours in the presence of Brefeldin (10 µg/ml). After surface staining and fixation with 1% PFA overnight at 4 \u003csup\u003eo\u003c/sup\u003eC, the cells were permeabilized by saponin, and antibodies for the intracellular staining were added.\u003c/p\u003e\u003cp\u003eFlow cytometry measurements were performed using an LSRFortessa (BD Bioscience, 5-laser configuration, Model No. 647794E6). The flow cytometry data were analyzed using FlowJo (BD Live Sciences, v. 10.7.1).\u003c/p\u003e\u003cp\u003eWe defined the cell types by the following marker (gating strategy in Fig S3): B cells (CD19\u003csup\u003e+\u003c/sup\u003e, CD3-), neutrophils (CD3\u003csup\u003e−\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e−\u003c/sup\u003e, Ly6C\u003csup\u003e+\u003c/sup\u003e, Ly6G\u003csup\u003e+\u003c/sup\u003e), classical monocytes (CD3\u003csup\u003e−\u003c/sup\u003e, CD9\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e−\u003c/sup\u003e, Ly6G\u003csup\u003e−\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003ehigh\u003c/sup\u003e), Ly6C\u003csup\u003elow\u003c/sup\u003e monocytes (CD3\u003csup\u003e−\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e−\u003c/sup\u003e, Ly6G\u003csup\u003e−\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003elow\u003c/sup\u003e), NK cells (CD3\u003csup\u003e−\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e+\u003c/sup\u003e), NK T cells (CD3\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e−\u003c/sup\u003e, CD4\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e+\u003c/sup\u003e), macrophages (CD3\u003csup\u003e−\u003c/sup\u003e, CD9\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e−\u003c/sup\u003e, Ly6G\u003csup\u003e−\u003c/sup\u003e, CD11b\u003csup\u003e+\u003c/sup\u003e, Ly6C\u003csup\u003elow\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003e), cDC1 (CD3\u003csup\u003e−\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e−\u003c/sup\u003e, CD88\u003csup\u003e−\u003c/sup\u003e, CD26\u003csup\u003e+\u003c/sup\u003e, CD64\u003csup\u003e−\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003e+\u003c/sup\u003e, XCR1\u003csup\u003e+\u003c/sup\u003e), cDC2 (CD3\u003csup\u003e−\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, NK1.1\u003csup\u003e−\u003c/sup\u003e, CD88\u003csup\u003e−\u003c/sup\u003e, CD26\u003csup\u003e+\u003c/sup\u003e, CD64\u003csup\u003e−\u003c/sup\u003e, MHCII\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003e+\u003c/sup\u003e, XCR1\u003csup\u003e−\u003c/sup\u003e) CD4 T cells (CD3\u003csup\u003e+\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, γδ TCR\u003csup\u003e−\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e−\u003c/sup\u003e), CD8 T cells (CD3\u003csup\u003e+\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, γδ TCR\u003csup\u003e−\u003c/sup\u003e, CD8\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e−\u003c/sup\u003e), γδ T cells (CD3\u003csup\u003e+\u003c/sup\u003e, CD19\u003csup\u003e−\u003c/sup\u003e, γδ TCR\u003csup\u003e+\u003c/sup\u003e) Th17 cell (CD3\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e−\u003c/sup\u003e, IFNγ\u003csup\u003e−\u003c/sup\u003e, IL-17\u003csup\u003e+\u003c/sup\u003e), Th1 cell (CD3\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e−\u003c/sup\u003e, IFNγ\u003csup\u003e+\u003c/sup\u003e, IL-17\u003csup\u003e−\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eRNA isolation and qPCR\u003c/p\u003e\u003cp\u003eCells were lysed using Tri reagent (Bio\u0026amp;Sell, order No: BS67.211.0100). The RNA was isolated using the Direct-zol™ -96 RNA isolation Kit (ZymoResearch, cat. No.: R2056) according to the instructions of the producer. The RNA was transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat No.: 4368814).\u003c/p\u003e\u003cp\u003eTo perform the qPCR, primers and probes from the KiCqStart® Probe Assay (Sigma-Aldrich) were used for \u003cem\u003eClec4e (Mincle)\u003c/em\u003e and \u003cem\u003eHprt\u003c/em\u003e (sequence see Table S2). For \u003cem\u003eIl6\u003c/em\u003e and \u003cem\u003eIl1b\u003c/em\u003e, the primers were used together with a probe from the Roche UPL universal probe library (sequence see Table S2). Gene expression was normalized using the \u003cem\u003eHprt\u003c/em\u003e expression, and the fold change was calculated using the 2\u003csup\u003e−ΔΔct\u003c/sup\u003e method \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, with isopropanol-treated samples serving as calibrators.\u003c/p\u003e\u003cp\u003eSerum collection and H1-specific Ab ELISA\u003c/p\u003e\u003cp\u003eBlood was collected at the end of the experiment by retroorbital bleeding from unconscious animals after i.p. application of Ketamin (100 µg/g bodyweight) together with Xylazin (10 µg/g bodyweight). Afterwards, still under narcosis, the mice were killed by cervical dislocation. Sera were separated from the blood by centrifugation (900 g, 10 min). H1 (400 ng/ml) was adsorbed overnight onto an ELISA plate (Sarstedt, ref: 82.1581.200). The non-bound H1 was discarded, and after BSA block (1% BSA in PBS), the sera were applied at different dilutions. The sera were washed away and the bound H1-specific Abs were detected using the following HRP-coupled secondary Ab: goat anti-mouse IgG1 (Southern Biotech, 1071-05, 1:4000), goat anti-mouse IgG2c (Southern Biotech, 1078-05, 1:4000), goat anti-mouse IgG2b (Southern Biotech, 1091-05, 1:4000).\u003c/p\u003e\u003cp\u003eStatistics\u003c/p\u003e\u003cp\u003eFor statistical analysis, GraphPad Prism (GraphPad Software, LLC, version 9.5.1) was used. The used statistical test for every graph is indicated in the figure legends. In all graphs, each dot corresponds to one mouse, the height of the bar indicates the mean. For the two-way ANOVA tests the normal distribution of the data was tested using the Shapiro-Wilk test. If the requirement of the normal distribution was not meet, the ANOVA was performed on log transformed values, this is indicated in the figure legend.\u003c/p\u003e\u003cp\u003eThe analysis of Ab titers was only done for immunized groups. To this end, a correlation was modeled between the measured OD values and the respective dilution step. For this, a three-parameter non-linear regression with the equation \u003cem\u003eOD = Bottom + (Top-Bottom)/(titer/mid-titer point)\u003c/em\u003e was used. The mid-titer point was obtained together with its SEM, and to compare these values between different groups, a one-way ANOVA followed by Dunnett’s multiple comparison test was performed (or Mann-Whitney U test in case of two groups).\u003c/p\u003e\u003cp\u003eThe p-values of all tests are displayed on the respective comparisons; to maintain clarity, only meaningful comparisons are shown in the graphs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Lars Nitschke, Kai Hildner, and David Vöhringer for providing different Cre-deleter mouse lines. We thank Drs. Gerhard Krönke and Jürgen Rech for supplying Etanercept. \u003c/p\u003e\n\u003cp\u003eMany thanks to Dr. Dieter Engelkamp and Martina Döhler for IVF of TNFR1fl mice. \u003c/p\u003e\n\u003cp\u003eAnimal husbandry by Manfred Kirsch and the PETZ staff is highly appreciated. Many thanks to Barbara Bodendorfer for her technical support. \u003c/p\u003e\n\u003cp\u003eThe work was done as part of the doctoral thesis of R. B..\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by Deutsche Forschungsgemeinschaft grants RTG 2599 “Fine Tuners of the Adaptive Immune Response” and LA 1262/8-1 to R.L. . U.S. was supported by RTG 2740 \"ImmunoMicroTope\" project A6. \u003c/p\u003e\n\u003cp\u003eThe funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eR.B. performed all experiments, except the IL-17 fate-mapping mouse experiment, analysed and visualized the data, drafted and revised the manuscript. C.H. contributed preliminary analysis in Mincle tg mice. G.K.P. performed the IL-17 fate-mapping mouse experiment, discussed results, and revised the manuscript. I.R., U.S., B.U.S., S.Y. provided mice or critical reagents, discussed results, and revised the manuscript. R.L. designed and supervised the study and drafted and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eDisclosure and competing interest statement\u003c/p\u003e\n\u003cp\u003eI.R. is coinventor on a patent of the cationic adjuvant formulations (CAF). All rights have been assigned to Statens Serum Institut, a Danish not-forprofit governmental institute. \u003c/p\u003e\n\u003cp\u003eAll other authors declare no financial or non-financial competing interests.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFischer, R., Kontermann, R. \u0026amp; Maier, O. Targeting sTNF/TNFR1 Signaling as a New Therapeutic Strategy. \u003cem\u003eAntibodies \u003c/em\u003e\u003cstrong\u003e4, \u003c/strong\u003e48\u0026ndash;70; 10.3390/antib4010048 (2015).\u003c/li\u003e\n\u003cli\u003eDavis, J. S., Ferreira, D., Paige, E., Gedye, C. \u0026amp; Boyle, M. 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A new mathematical model for relative quantification in real-time RT-PCR. \u003cem\u003eNucleic acids research \u003c/em\u003e\u003cstrong\u003e29, \u003c/strong\u003ee45; 10.1093/nar/29.9.e45 (2001).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7495259/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7495259/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSuccessful induction of protective T cells by subunit vaccines requires adjuvants. The adjuvant CAF01 potently induces robust Th17 responses that depend on the C-type lectin receptor Mincle and TNF. Mincle expression is low in resting macrophages, but upregulated by TNF. Here, we used conditional TNFR1-deficient mice to dissect cell type-specific contributions of TNF signaling to Th17 induction by the recombinant tuberculosis fusion protein H1 adjuvanted with CAF01. TNFR1 in myeloid cells was essential, whereas TNFR1 deletion in DC only partially reduced Th17 cells, and TNFR1 was not required in T cells. Constitutive, TNF-independent transgenic Mincle expression restored Th17 induction despite TNF blockade. Thus, regulation of Mincle by TNF plays a causal role, likely by controlling production of Th17-polarizing cytokines in monocytes. Together, we show that induction of Th17 by CAF01 requires TNF signaling in myeloid cells, with enhanced adjuvant sensing due to Mincle upregulation as a potential mechanism.\u003c/p\u003e","manuscriptTitle":"TNF promotes Th17 vaccine responses by enabling myeloid cell pattern recognition via Mincle","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 10:33:11","doi":"10.21203/rs.3.rs-7495259/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a7ae761f-16e2-439a-9b83-35b329336e77","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55283846,"name":"Biological sciences/Cell biology"},{"id":55283847,"name":"Biological sciences/Immunology"}],"tags":[],"updatedAt":"2025-10-22T01:23:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 10:33:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7495259","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7495259","identity":"rs-7495259","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}