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. 2016 Jul;9(4):894-906.
doi: 10.1038/mi.2015.118. Epub 2015 Nov 18.

Colonic tolerance develops in the iliac lymph nodes and can be established independent of CD103(+) dendritic cells

S Veenbergen  1 , L A van Berkel  1 , M F du Pré  1 , J He  2 , J J Karrich  3 , L M M Costes  1 , F Luk  1 , Y Simons-Oosterhuis  1 , H C Raatgeep  1 , V Cerovic  4 , T Cupedo  3 , A M Mowat  5 , B L Kelsall  2 , J N Samsom  1
Affiliations

Colonic tolerance develops in the iliac lymph nodes and can be established independent of CD103(+) dendritic cells

S Veenbergen et al. Mucosal Immunol. 2016 Jul.

Abstract

Tolerance to harmless exogenous antigens is the default immune response in the gastrointestinal tract. Although extensive studies have demonstrated the importance of the mesenteric lymph nodes (MLNs) and intestinal CD103(+) dendritic cells (DCs) in driving small intestinal tolerance to protein antigen, the structural and immunological basis of colonic tolerance remain poorly understood. We show here that the caudal and iliac lymph nodes (ILNs) are inductive sites for distal colonic immune responses and that colonic T cell-mediated tolerance induction to protein antigen is initiated in these draining lymph nodes and not in MLNs. In agreement, colonic tolerance induction was not altered by mesenteric lymphadenectomy. Despite tolerance development, CD103(+)CD11b(+) DCs, which are the major migratory DC population in the MLNs, and the tolerance-related retinoic acid-generating enzyme RALDH2 were virtually absent from the ILNs. Administration of ovalbumin (OVA) to the distal colon did increase the number of CD11c(+)MHCII(hi) migratory CD103(-)CD11b(+) and CD103(+)CD11b(-) DCs in the ILNs. Strikingly, colonic tolerance was intact in Batf3-deficient mice specifically lacking CD103(+)CD11b(-) DCs, suggesting that CD103(-) DCs in the ILNs are sufficient to drive tolerance induction after protein antigen encounter in the distal colon. Altogether, we identify different inductive sites for small intestinal and colonic T-cell responses and reveal that distinct cellular mechanisms are operative to maintain tolerance at these sites.

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Figures

Figure 1
Figure 1. Different inductive sites for small intestinal and colonic T-cell responses
(a) Schematic illustration of the location of colon-draining ILN. (b, c) BALB/c mice received 3.5 mg OVA orally (i.g.) or 1.7 mg OVA intracolonically (i.c.), either labeled with Alexa Fluor-488 succinimidyl ester or unlabeled. Twenty hours after OVA administration, MLN and ILN were digested using liberase/DNAse and single cell suspensions were stained for CD11c and analyzed for Alexa-Fluor-488+ cells by flow cytometry. (b) Representative dot plots and (c) percentage CD11chighOVA-488 positive cells determined by flow cytometry are shown. Results are depicted as mean plus SEM and are representative of two independent experiments using 3 mice per experiment. **P<0.01 versus control, by Mann-Whitney U test. (d, e) CD4+KJ1.26+ OVA-specific T cells were purified from DO11.10 transgenic × Rag−/− mice and labeled with CFSE. Subsequently, BALB/c mice were given 6 × 106 CFSE-labeled T cells intravenously and one day later, received 70 mg OVA either i.g. or i.c. 72h after OVA administration, MLN and ILN were analyzed for antigen-specific T-cell proliferation by measuring CFSE dye dilution. (d) Histogram plots of CFSE fluorescence of OVA-specific TCR-transgenic CD4+ T cells and (e) percentage proliferating cells determined by flow cytometry are shown. The data shown are from two independent experiments and are depicted as mean plus SEM. ***P< 0.001 versus control, by Mann-Whitney U test. (f) Whole cell preparations of lymph nodes were analyzed for expression of Gpr15 and Ccr9 mRNA by quantitative PCR analysis. Values are mean plus SEM for 3 mice per group. *P< 0.05, **P<0.01 versus control, by Student's t-test.
Figure 2
Figure 2. Colonic OVA administration results in de novo Foxp3+ Treg induction and induces systemic tolerance
(a, b) CD4+KJ1.26+ OVA-specific T cells were purified from DO11.10 transgenic × Rag−/− mice and labeled with CFSE. Subsequently, BALB/c mice were given 6 × 106 CFSE-labeled T cells intravenously and one day later, received 70 mg OVA either i.g., i.c. or 400 μg OVA intramuscularly (i.m.). 72 h after OVA administration, draining lymph nodes (MLN for i.g., ILN for i.c. and popliteal/inguinal lymph nodes for i.m.) were isolated and analyzed for CD4+KJ1.26+CFSE+Foxp3+ T cells by flow cytometry. Values are depicted as mean plus SEM from 2 independent experiments with 4-8 mice per experimental condition. *P< 0.05, **P<0.01 versus control, by one-way ANOVA. (c) Mice were treated with saline, 25 mg OVA i.g. or i.c. and five days later were sensitized subcutaneously in the tail base with 100 μg OVA emulsified in Incomplete Freund's adjuvant. Five days after sensitization, mice were challenged with 10 μg OVA in the auricle of both ears. After 24 h, increases in ear thickness in both ears were determined and compared with values before challenge. Values are mean plus SEM for 5-7 mice per experimental condition. *P< 0.05, **P< 0.01 versus control, by one-way ANOVA. (d) Mice were adoptively transferred with 6 μ 106 CFSE-labeled CD4+KJ1.26+ T cells and one day later, received 70 mg OVA i.g., i.c. or 400 μg OVA i.m. 72 h after OVA administration, draining lymph nodes were isolated and enriched for CD4+ T cells. Subsequently, 1 × 105 CD4+KJ1.26+ T cells were transferred to naive second acceptor mice and one day after the transfer, recipient mice were sensitized subcutaneously in the tail base with 100 μg OVA emulsified in Incomplete Freund's adjuvant. After 5 days, mice were challenged with 10 μg OVA in the auricle of both ears. After 24h, increases in ear thickness in both ears were determined and compared with values before challenge. Data are mean plus SEM for 4-7 mice per experimental condition. *P< 0.05, **P< 0.01 versus control, by one-way ANOVA. (e, f) MLNs were surgically excised (MLNx) and after 6 weeks mice were treated with 25 mg OVA i.c. Three days later, mice were sensitized subcutaneously in the tailbase with 100 μg OVA emulsified in Incomplete Freund's adjuvant. At day 8, mice were challenged with 10 μg OVA in the auricle of both ears. (e) After 24h, increases in ear thickness in both ears were determined and compared with values before challenge. (f) Cells purified from the inguinal lymph nodes after induction of the DTH reaction were restimulated for 48h with OVA protein. IFNγ levels were determined in supernatant by ELISA. Completeness of mesenteric lymphadenectomy was confirmed in all MLNx animals at autopsy. The results are depicted as mean plus SEM for 5-7 mice per experimental condition. **P< 0.01 versus control, using one-way ANOVA.
Figure 3
Figure 3. Differential composition of DC subsets in MLN and ILN
Mice received 70 mg OVA i.g. or i.c. and 16-18h later, MLN and ILN were digested using liberase/DNAse and single cell suspensions were stained for CD45, F4/80, CD11c, MHCII, CD11b, CD103 and CCR7. (a,b) Live CD45+F4/80− single cells were gated and then analyzed for CD11c+MHCIIhigh migratory DCs. (a) Representative dot plots and (b) percentage CD11c+MHCIIhigh migratory DCs determined by flow cytometry are shown. The data shown are from three independent experiments (3 mice pooled for each experimental condition per experiment) and depicted as mean plus SEM. ***P< 0.001 versus control, by one-way ANOVA. (c) The CD11c+MHCIIhigh migratory DC population was further analyzed by assessing the expression of CD103 and CD11b. (d) Absolute cell numbers of CD103+CD11b− and CD103−CD11b+ DCs from ILN (upper panel) and CD103+CD11b+, CD103+CD11b− and CD103−CD11b+ DCs from MLN (lower panel) after OVA administration. The data shown are from three independent experiments using cells pooled from 2-3 mice for each experimental condition per experiment. *P< 0.05, **P< 0.01 ***P< 0.001 versus control, using one-way ANOVA. (e) Migratory DC subsets were stained for CCR7 after OVA i.g. (MLN) or i.c. (ILN). Representative histogram plots are shown of two independent experiments.
Figure 4
Figure 4. CD103+ DCs from the colon-draining lymph nodes express low RALDH2 levels
(a, b) MLN and ILN were digested using liberase/DNAse and single cell suspensions were stained for CD45, F4/80, CD11c, MHCII, CD11b, CD103. Live CD45+F4/80− single cells were gated and further analyzed for the expression of CD103 and CD11b within the CD11c+MHCIIhigh migratory DCs. The DC subsets were then analyzed for (a) IRF4 or (b) IRF8 expression by intracellular staining. The results shown are from one of two experiments using cells pooled from 8 mice. (c, e) CD103+ and CD103− DC subsets were purified by flow cytometric cell sorting and expression of (c) Ptgs2 (e) aldh1a2 mRNA was determined by quantitative PCR analysis. Values are depicted as mean plus SEM from 4 independent experiments using cells pooled from 20-30 mice per experiment. *P< 0.05 versus control, by one-way ANOVA. (d, f) Whole cell preparations of lymph nodes were analyzed for expression of (d) Ptgs2 and (f) aldh1a2 mRNA by quantitative PCR analysis. The peripheral lymph nodes (PLN) include the inguinal, axillary, brachial lymph nodes. Values are mean plus SEM for 3-7 mice per group. *P< 0.05, **P< 0.01 versus control, by one-way ANOVA.
Figure 5
Figure 5. Batf3−/− mice specifically lacking CD103+CD11b− DC display a normally suppressed DTH response after colonic OVA administration
(a) MLN and ILN were isolated from C57BL/6 and Batf3−/− mice and single cell suspensions were stained for CD45, F4/80, CD11c, MHCII, CD11b, and CD103. Live CD45+F4/80− single cells were gated and further analyzed for the expression of CD103 and CD11b within the CD11c+MHCIIhigh migratory DCs. (b) The percentage of CD4+Foxp3+ T cells in MLN and ILN from untreated C57BL/6 and Batf3−/− mice was determined by flow cytometry. Values are depicted as mean plus SEM for 6 mice (c, d) Naive CD4+ OVA-specific T cells were purified from OTII transgenic × Rag−/− mice and labeled with CFSE. At the same time, CD11c+ cells were isolated from MLN and ILN of C57BL/6 and Batf3−/− mice by flow cytometric cell sorting and loaded with OVA protein for 1 hour. OVA-loaded CD11c+ cells were co-cultured with CFSE-labeled OVA-specific naive T cells for 72 hours. (c) Histogram plots of CFSE fluorescence of OVA-specific TCR-transgenic CD4+ T cells and percentage proliferating cells in each division peak. (d) Foxp3+CFSE+ T cells amongst CD4+ T cells in the presence or absence of TGFβ determined by flow cytometry. The results are means plus SD from triplicate cultures using cells pooled from 33 Batf3−/− mice or 45 C57BL/6 mice. *P< 0.05 versus control, by Student's t test. (e, f) Mice were treated with saline, 25 mg OVA i.g. or i.c. and three days later were sensitized subcutaneously in the tailbase with 100 μg OVA emulsified in Complete Freund's adjuvant. At day 8, mice were challenged with 10 μg OVA in the auricle of both ears. (e) After 48h, increases in ear thickness in both ears were determined and compared with values before challenge and (f) CD4+ T cells were purified from the inguinal lymph nodes of Batf3−/− mice after induction of the DTH reaction and were cocultured with splenic CD11c+ cells from C56BL/6 wild-type mice and restimulated for 48h with OVA protein. IFNγ levels were determined in supernatant by ELISA. The results are depicted as mean plus SEM for 4 or 5 mice per experimental condition. *P< 0.05 versus control, using one-way ANOVA.
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