We examined the colons of mice lacking αvβ8 on DCs for the presence of IL-17–producing CD4+
T cells. Intracellular flow cytometry analysis of cells isolated from the colonic lamina propria of β8fl/fl
× CD11c-cre mice showed a near absence of IL-17–expressing CD4+
T cells (0.6% ± 0.2%), whereas 8.5% ± 1.3% of colonic CD4+
T cells in control mice expressed IL-17 (Figure , A and B). In contrast, in β8fl/fl
× CD11c-cre mice a majority (52% ± 4.4%) of CD4+
T cells expressed IFN-γ (Figure B). Since IFN-γ impedes development of Th17 cells in vitro, we examined whether increased production of IFN-γ was responsible for impaired Th17 development in β8fl/fl
× CD11c-cre mice by crossing them to mice deficient in IFN-γ. The colons of Ifng–/–
× CD11c-cre mice also showed a dramatic reduction in the number of CD4+
cells, with only 3.1% ± 0.5% of the CD4+
cells expressing IL-17 compared with 26% ± 4.0% in control Ifng–/–
mice (Figure , C and D). Interestingly, Ifng–/–
× CD11c-cre mice exhibited colitis and enlarged mesenteric lymph nodes that closely resembled β8fl/fl
× CD11c-cre mice, suggesting that the colitis seen in the absence of αvβ8 on DCs occurs independently of IFN-γ (data not shown). We previously reported that β8fl/fl
× CD11c-cre mice have a decreased frequency of Tregs in the colon but a normal proportion in the spleen (14
). Since Tregs are significant producers of TGF-β, we questioned whether a decreased frequency of Tregs in the colon could decrease the amount of total TGF-β available for Th17 development in β8fl/fl
× CD11c-cre mice. We found no significant difference in the amount of TGF-β in the colons from control and β8fl/fl
× CD11c-cre mice (417 ± 29 and 384 ± 13 pg/ml; P
= 0.16). These data indicate that excess production of IFN-γ and total TGF-β concentrations were not responsible for the absence of Th17 cells in the colons of β8fl/fl
× CD11c-cre mice.
Impaired Th17 development in β8fl/fl × CD11c-cre mice.
To determine the potential clinical relevance of impaired IL-17 production by T cells stimulated with αvβ8-deficient DCs, we employed the EAE disease model. The EAE model has been extensively studied and depends on the formation of pathogenic IL-17–producing T cells. We immunized control and β8fl/fl × CD11c-cre mice for EAE and scored them based on paralysis severity. β8fl/fl × CD11c-cre mice were almost completely protected from paralysis (3.1 ± 0.2 mean peak EAE clinical score in control mice vs. 0.2 ± 0.1 in β8fl/fl × CD11c-cre mice; Figure A). We then examined the CNS of mice at peak disease for the presence of Th17 cells. As expected, induction of EAE caused a marked accumulation of CD4+ and CD8+ T cells in the CNS of control mice, but the numbers of T cells were dramatically reduced in the CNS of β8fl/fl × CD11c-cre mice (Figure B). Of the CD4+ T cells that were present in the CNS of in β8fl/fl × CD11c-cre mice, 1.4% ± 0.4% expressed IL-17 (Figure , C and D). In contrast, 23% ± 2% of CD4+ T cells produced IL-17 in the CNS of control mice (Figure , C and D).
β8fl/fl × CD11c-cre mice are protected from EAE.
Since Th17 cells were nearly absent in the CNS of β8fl/fl
× CD11c-cre mice and these mice also exhibited a marked reduction in the total number of CNS T lymphocytes, we examined the periphery of these mice for Th17 cells before onset of EAE. At day 8 after immunization for EAE, we observed 9.2% ± 3% of CD4+
T cells producing IL-17 in the draining lymph nodes of control mice, but only 0.6% ± 0.1% in the draining lymph nodes of β8fl/fl
× CD11c-cre mice (Figure , E and F). To determine whether mice lacking integrin αvβ8 on DCs could develop EAE if the T cell priming phase was bypassed, we transferred activated 2D2 TCR transgenic T cells (transgenic for a T cell receptor to the MOG35–55
peptide derived from myelin oligodendrocyte protein) into control and β8fl/fl
× CD11c-cre mice. Control and β8fl/fl
× CD11c-cre mice developed similar EAE after passive immunization with activated 2D2 T cells (Supplemental Figure 1; supplemental material available online with this article; doi:
). We also checked whether β8 expression on microglia, an important antigen-presenting cell in the CNS, was affected in β8fl/fl
× CD11c-cre mice. We found no difference in microglia β8 mRNA expression from control and β8fl/fl
× CD11c-cre mice (1.3 × 10–3
± 4.2 × 10–4
and 1.1 × 10–3
± 2.5 × 10–4
= 0.35). These data support a critical role for induction of Th17 cells by αvβ8 on DCs in periphery during the development of EAE.
To assess whether integrin αvβ8 on DCs participates in the conversion of naive CD4+ T cells to Th17 cells, we transferred Thy1.1-labeled naive CD4+ 2D2 TCR transgenic T cells into control and β8fl/fl × CD11c-cre mice immunized with an emulsion of MOG35–55 and CFA. The Thy1.1 label allowed us to identify the transferred cells among endogenous CD4+ T cells upon re-isolation from the draining lymph nodes and spleen. When transferred Thy1.1+ 2D2 T cells were re-stimulated and analyzed 8 days after immunization, 60% ± 1.1% of the cells produced IL-17 (Figure , A and B). However, only 22% ± 1.4% Thy1.1+ 2D2 T cells produced IL-17 after transfer into β8fl/fl × CD11c-cre mice (Figure , A and B). Additionally, IL-17–expressing 2D2 T cells recovered from β8fl/fl × CD11c-cre mice produced significantly less IL-17 as measured by MFI of IL-17 staining (Figure C). We also determined the role of integrin αvβ8 on DCs in the conversion of naive CD4+ T cells to Th17 cells by transferring polyclonal naive CD4+ T cells into β8fl/fl × CD11c-cre mice crossed onto the SCID background. SCID mice lack functional T and B cells and therefore allowed us to study the role of integrin αvβ8 on DCs in Th17 differentiation in the absence of endogenous lymphocytes. When naive CD4+ T cells were transferred into SCID control mice and recovered from the colonic lamina propria and spleen 12 days later, 10% ± 1.2% and 16.1% ± 2.4%, respectively, produced IL-17 upon restimulation (Figure , D and E). However, only 1.2% ± 0.3% and 2.8% ± 0.6% of CD4+ T cells recovered from the colonic lamina propria and spleen, respectively, of SCID β8fl/fl × CD11c-cre mice produced IL-17. Furthermore, the few IL-17–expressing CD4+ T cells isolated from SCID β8fl/fl × CD11c-cre mice produced significantly less IL-17 as measured by MFI of IL-17 staining (Figure F). These data indicate that integrin αvβ8 on DCs is critical for the development of Th17 cells in response to antigen challenge in vivo.
Blunted Th17 conversion of naive CD4+ T cells adoptively transferred into β8fl/fl × CD11c-cre mice.
To examine the mechanism by which loss of αvβ8 on DCs protected mice from EAE and impaired Th17 differentiation, we characterized DCs from the draining lymph nodes of β8fl/fl × CD11c-cre mice. DCs have been categorized into the lymphoid, myeloid, and plasmacytoid groups based on their developmental origin and function. The presence of these DC populations in the draining lymph nodes of control or β8fl/fl × CD11c-cre mice was determined 7 days after immunization for EAE. We observed 31.4% ± 1.7% lymphoid, 66.8% ± 1.7% myeloid, and 3.9% ± 0.6% plasmacytoid DCs in control mice and 35.1% ± 3.4% lymphoid, 62.6% ± 3.4% myeloid, and 4.5% ± 0.6% plasmacytoid DCs in β8fl/fl × CD11c-cre mice (Figure , A and B). Next, we checked the ability of DCs from β8fl/fl × CD11c-cre mice to stimulate the proliferation of naive T cells after immunization for EAE. At day 7 after immunization, there were 6.0% ± 1.5% myelin oligodendrocyte glycoprotein–specific (MOG-specific) T cells after enrichment for tetramer-labeled cells in control mice and 8.1% ± 0.8% in β8fl/fl × CD11c-cre mice (Figure , C and D). Furthermore, we saw that MOG-specific TCR transgenic T cells (clone 2D2) adoptively transferred into control and β8fl/fl × CD11c-cre mice underwent a similar number of cell divisions following EAE immunization (Figure , E and F). We then analyzed the expression of costimulatory and coinhibitory molecules on DCs from β8fl/fl × CD11c-cre mice. As shown in Table , no significant differences were found in the expression of CD40, CD80, CD86, PD-L1, PD-L2, ICAM-1, OX40L, ICOSL, or MHCII. Finally, we examined the ability of DCs from β8fl/fl × CD11c-cre mice to produce cytokines related to the development of Th17 cells. As shown in Table , we found no significant differences in the levels of Il1b, Il6, Il23, Tgfb, or Tnfa mRNA or secreted protein. We also measured the effects of CFA immunization on DC β8 integrin expression in control mice and found that CFA slightly reduced β8 mRNA, from 5.0 × 10–5 ± 7.3 × 10–6 to 2.4 × 10–5 ± 3.2 × 10–6 (P = 0.037). Collectively, these data suggest that DCs from β8fl/fl × CD11c-cre mice are similar to control mice in their composition, ability to stimulate naive CD4 T cell proliferation, and capacity to make cytokines that promote Th17 differentiation.
Characterization of draining lymph node DCs in β8fl/fl × CD11c-cre mice.
Costimulatory and coinhibitory protein expression on DCs from β8fl/fl × CD11c-cre mice
Production of Th17-promoting cytokines by DCs from β8fl/fl × CD11c-cre mice
Integrin αvβ8 controls the activation of TGF-β1 and -β3 through interaction with an Arg-Gly-Asp sequence on the LAP of the latent TGF-β complex (11
). To determine whether integrin αvβ8 on DCs regulates Th17 development through activation of latent TGF-β, we used an in vitro assay to monitor Th17 development in the presence of control or αvβ8-deficient DCs. As shown in Figure , αvβ8-deficient DCs cultured with naive CD4+
2D2 T cells under conditions that drive Th17 development (i.e., IL-1β and IL-6), but in the absence of exogenous TGF-β, showed a reduced ability to promote IL-17 production by T cells. However, IL-17 production after culture with αvβ8-deficient DCs was restored by the addition of exogenous TGF-β (Figure , A–C). Furthermore, the addition of a TGF-β–neutralizing antibody significantly reduced IL-17 production by T cells cultured with control DCs but had a minimal effect on αvβ8-deficient DCs (Figure , A–C). To confirm that IL-17 produced by T cells in this assay was from bona fide Th17 cells, we measured IL-17F, another canonical Th17 cytokine. Indeed, IL-17F was detected in the supernatant of cultures grown without the addition of exogenous TGF-β and was significantly reduced in cultures with αvβ8-deficient DCs (Figure D). Furthermore, we measured mRNA expression of 2 genes that have been shown previously to be regulated by TGF-β — Rorc
, which encodes the Th17-specific transcription factor RORγt, and Il23r
, which encodes the IL-23 receptor, a receptor required for clonal expansion of Th17 cells. We detected transcripts of these genes in T cells from cultures with control DCs but found a dramatic reduction in expression of these genes in T cells from cultures with αvβ8-deficient DCs (Figure E).
DCs from β8fl/fl × CD11c-cre mice fail to drive Th17 development through a defect in TGF-β activation.
The mechanism by which integrin αvβ8 on DCs presents active TGF-β to induce IL-17 expression in T cells is unknown. One possibility is that latent TGF-β is constitutively activated by αvβ8 on DCs to create a surrounding milieu of active TGF-β (i.e., a field effect). Alternatively, αvβ8 might only induce IL-17 expression in T cells if it activates TGF-β in the context of antigen presentation. This would be a teleologically attractive mechanism to tightly regulate, in space and time, this important immunologic decision. To distinguish between these mechanisms, we cultured naive CD4+ 2D2 T cells under Th17-polarizing conditions (in the absence of exogenous TGF-β) with mixtures of αvβ8-deficient DCs and αvβ8-containing DCs that express either matched or mismatched MHCII. Thus, MHCII-mismatched DCs would be unable to present MOG peptides to 2D2 T cells. We first established that a 1:1 mixture of control and αvβ8-deficient DCs was able to completely rescue the defect in IL-17 induction seen with αvβ8-deficient DCs alone (Figure ). We then cultured αvβ8-expressing, MHCII-mismatched (H2q MHC haplotype from FVB mice) DCs at a 4:1 mixture with αvβ8-deficient DCs (H2b MHC haplotype from C57BL/6 mice). MHCII-mismatched DCs were unable to rescue the impaired IL-17 production seen with αvβ8-deficient DCs (Figure ). These data suggest that Th17 cell induction requires simultaneous presentation of antigen (by MHCII) and activation of TGF-β by integrin αvβ8 on the same DCs.
Th17 development through integrin αvβ8 on DCs is dependent on MHCII-TCR engagement.