(A) TaqMan qPCR for Aldh1a1 and Aldh1a2 mRNA expression in DC (encoding for Raldh1 and Raldh2, respectively) (n=4). Results were normalized respect to Actb mRNA (encoding β-actin) (B) Raldh activity (aldefluor assay) in DC either in the absence or the presence of the Raldh inhibitor DEAB (n=4). (C) Naïve CFSE-labeled P14xTCRα^-/- or OT-IxRAG2^-/- CD8 T cells were activated with peptide-pulsed DC from peripheral lymph nodes (PLN-DC), mesenteric lymph nodes (MLN-DC), Peyer's patches (PP-DC) or small intestine lamina propria (LP-DC) from wild type or MyD88^-/- mice. After 4-5 days T cells were analyzed for α4β7 and CCR9 expression by flow cytometry (n=6). Numbers indicate percentage of positive cells and in parenthesis the mean fluorescence intensity (MFI) of total cells. (D-E) Wild type CFSE-labeled OT-1xRAG2^-/- CD8 T cells (Thy1.1⁺) were adoptively transferred into wild type or MyD88^-/- mice (Thy1.2⁺), which were immunized i.p. with ovalbumin plus Alum. 4 days post-immunization the mice were analyzed for (D) the expression of α4β7 and CCR9 on transferred activated CFSE^Low CD8 T cells in PLN, MLN (n=4) and (E) the numbers of Thy1.1⁺ CD8 T cells recovered from the small bowel lamina propria (LP) and intraepithelial lymphocyte compartment (IEL) or the spleen (n=6-8). (F) Same as in (D), but the mice were immunized orally with ovalbumin (n=4). Mean ± SEM. * p<0.05, **p<0.01, ***p<0.001

(A) α4β7 and CCR9 staining on CFSE-labeled P14xTCRα^-/-CD8 T cells activated by CD11c⁺ DC from BM chimeras in which BM from either wild type or MyD88^-/- donors was transplanted into wild type or MyD88^-/- recipient mice (n=3). (B-D) Mixed BM chimeras were generated by transplanting irradiated wild type mice with a 1:1 ratio of BM from MyD88-/- mice plus BM from CD11c-DTR mice. (B, C) 10 weeks later the mice were treated with diphtheria toxin (DT), which depleted CD11c⁺ DC derived from CD11c-DTR BM. (B) MLN-DC were analyzed for their Raldh activity (n=7). (C) Alternatively, the chimeric mice were adoptively transferred with OT1 CD8 T cells and then immunized orally with ovalbumin. Four days later the activated CD8 T cells were analyzed for their expression of α4β7 and CCR9 in MLN (n=5). (D) MyD88^-/- and MyD88-sufficient CD103⁺ MLN-DC from chimeric mice (without DT treatment) were analyzed for their Raldh activity (n=2). Mean ± SEM. * p<0.05

(A) TLR mRNA expression in freshly isolated DC. Representative of two experiments with similar results. (B) Aldh1a2 mRNA expression in wild type or MyD88^-/- Spleen-DC untreated (UT) or pre-treated with the indicated TLR ligands (n=3-6). (C) Raldh activity in DC. Numbers indicate percentage of aldefluor positive cells in the indicated gate (n=5). (D) α4β7 and CCR9 expression on CD8 T cells activated with Spleen-DC untreated (UT) or pre-treated with Pam₃CSK₄ (1 μg/ml) (n=6). Flow cytometry plots show percentage of positive cells and in parenthesis MFI of total cells. (E) α4β7 expression on T cells activated with Spleen-DC UT or pre-treated with Pam₃CSK₄ in FBS-free media with or without 50 nM retinol or 1 μM LE540 (RAR antagonist) (n=3). (F) Spleen-DC UT or pre-incubated for 24h with E. coli (K12 strain) and then analyzed for their expression of Aldh1a2 mRNA (n=2) or used to activate naïve CD8 T cells. T cells were analyzed after 4 days for their expression of α4β7 (n=3). (G) P14xTCRα^-/- CD8 T cells activated with Spleen-DC UT or pre-treated with Pam₃CSK₄ were labeled with TRITC or CFSE, respectively, mixed in a 1:1 ratio and injected i.v. into congenic Thy-1.1⁺ recipient mice. After 18h the ratio of CFSE⁺ to TRITC⁺ cells was analyzed in the indicated tissues. The homing indices (HI) were calculated as the ratio of CFSE⁺/TRITC⁺ in each tissue divided by the input ratio (n= 4). Statistics were calculated using a one sample t-test versus a HI=1 (equal migration). (H) Thy-1.1⁺ mice were adoptively transferred with naïve CFSE-labeled P14xTCRα^-/-CD8 T cells and then immunized i.v. with LCMVgp_33-41-pulsed Spleen-DC either UT or pre-treated with Pam₃CSK₄. Three days later α4β7 expression was analyzed on Thy1.2⁺ CD8 T cells in PLN, MLN, and spleen (n=3). (I) Human monocyte-derived DC (moDC) were UT or pre-treated with Pam₃CSK₄ and then analyzed for their expression of ALDH1A2 mRNA (n=4) or co-cultured with human T cells activated with plate-bound anti-CD3 plus anti-CD28 antibodies. T cells were analyzed after 5 days for their expression of α4β7 and CCR9 (n=3). Mean ± SEM. * p<0.05, ** p<0.01, ***p<0.001

(A) Raldh activity (aldefluor assay) in CD103⁺ MLN-DC from wild type or TLR2^-/-untreated (non-Flt3L DC expanded) mice (n=8 mice/group). (B) Naïve OT-IxRAG2^-/- CD8 T cells were activated with peptide-pulsed PLN-DC, MLN-DC or PP-DC from wild type or TLR2^-/- mice. After 4 days T cells were analyzed for α4β7 and CCR9 expression (n=4). Numbers indicate percentage of positive cells. (C-D) Wild type CFSE-labeled OT-1xRAG2^-/- CD8 T cells (Thy1.1⁺) were adoptively transferred into wild type or TLR2^-/- mice (Thy1.2⁺), which were immunized i.p. with ovalbumin plus LPS. (C) Four days post-immunization the mice were analyzed for the expression of α4β7 and CCR9 on activated Thy1.1⁺ CD8 T cells in MLN (n=5). (D) Numbers of Thy1.1⁺ CD8 T cells recovered from the small bowel lamina propria (LP) and intraepithelial lymphocyte compartment (IEL) 4 days post-immunization. (E) Raldh activity in CD103⁺ MLN-DC from wild type or TLR6^-/- untreated (non-Flt3L DC expanded) mice (n=2 mice/group). Mean ± SEM. * p<0.05, ***p<0.001

(A) Spleen-DC were untreated (UT) or pre-treated for 24 h with 1 μg/ml Pam₃CSK₄ plus/minus pharmacological inhibitors of p38/MAPK (10 μM SB203580), ERK/MAPK (10 μM FR180204), JNK/MAPK (50 μM SP600125) or NF-kB (50 μM SN50). After that DC were washed, pulsed with peptide and used to activate naïve CFSE-labeled OT-IxRAG2^-/- or P14xTCRα^-/- CD8 T cells. Four days later T cells were analyzed for their expression of α4β7 and CCR9 (n=6). (B-D) Spleen-DC were UT or pre-treated with Pam₃CSK₄ plus/minus 50 μM SP600125 (JNK inhibitor) or 1 μM LE540 (RAR antagonist). (B) Aldh1a2 mRNA expression in DC (n=3). (C) Raldh activity (aldefluor staining) in DC (n=3). (D) Luciferase activity in Spleen-DC from DR5-luciferase mice (n=3). Blockade of RA activity using 1 μM LE540 (RAR antagonist) was used as control. (E) Spleen-DC from wild type or JNK1^-/- mice were untreated (UT) or pre-treated for 24 h with 1 μg/ml Pam₃CSK₄ plus/minus 50 μM SP600125. After that DC were washed, pulsed with peptide and used to activate naïve CFSE-labeled OT-IxRAG2^-/- CD8 T cells. Four days later T cells were analyzed for their expression of α4β7 and CCR9 (n=3). (F) Wild type mice were orally treated with 50 μg/g SP600125 i.p. once a day for 5 days. After that, CD11c⁺ MLN-DC were analyzed for CD103 expression and Raldh activity (n=4 mice/group). Mean ± SEM. * p<0.05, ***p<0.001