Supplementary Methods
Mice and reagents. Young adult C57BL/6 mice were purchased from Jackson Laboratories. T-GFP
mice expressing GFP in naïve T cells and P14xT-GFP double transgenic mice have been described 1.
OT-IxRAG-/- mice, OVA-derived SIINFEKL peptide, and Flt-3L transfectants were provided by Dr. H.
Ploegh (Harvard Medical School). Mice were housed in a SPF/VAF facility and used between 8-16
weeks of age in accordance with NIH guidelines. MAbs and cytokine staining kits were purchased from
Pharmingen. LCMV gp33-41 peptide (KAVYNFATC, >95% purity, reconstituted at 1 mg/ml in sterile
water and stored at –80oC) was from Biosource. Murine rIL-2 (R&D Systems) was reconstituted to 20
g/ml in sterile PBS and stored at –80oC. Chemokines (R&D Systems) were reconstituted to 10 M in
sterile PBS and stored at –80oC.
DC isolation. Female C57BL/6 mice (6-8 weeks old) were injected subcutaneously with 107 B16
melanoma cells secreting Flt3-L 2. After 12 to 17 days, mice were sacrificed and PP, PLN, and spleens
were digested using 400 U/ml collagenase-D (Roche) plus 100 g/ml DNAse-I (Roche) for 40 min at
37oC with mild agitation. Single cell suspensions were generated from digested tissues and DC were
immunomagnetically isolated by negative selection 3. Briefly, 108 cells/ml were incubated for 20 min at
4oC with 10 g/ml MAbs to B220 (RA3-6B2), CD3 (17A2), and 5 g/ml Mabs to CD19 (1D3), Pan-NK
(DX5), Ly-76 (Ter-119), Ly-6G (Gr-1), and Thy-1 (G7). After washing, cells were incubated for 10
min with 80 l/ml goat anti-rat IgG microbeads (Miltenyi Biotec). Cells were washed and negatively
selected with CS negative selection columns (Miltenyi Biotec) following the manufacturer’s
instructions. Cell separation was performed at 4oC in PBS containing 2 mM EDTA and 2% FBS (Gibco
BRL, Australian, low endotoxin). Negatively selected DC (85-90% CD11c+) were resuspended to
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107/ml and pulsed for 2 hours at 37oC with 10 g/ml gp33-41 or SIINFEKL peptide in IMDM plus 10%
FBS and supplemented with 3 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 50 g/ml
gentamycin, 0.5 g/ml fungizone, and 50 M ß-mercaptoethanol (complete IMDM). Gentamycin,
fungizone, and ß-mercaptoethanol, were added immediately before use (a 50 mM stock solution of ßmercaptoethanol was prepared every 3 month in PBS and stored at 4oC). Pulsed DC were washed twice
at 37oC and used immediately in co-cultures. For some experiments, negatively selected DC were
stained with FITC-anti-CD11c (HL3) and a cocktail of phycoerythrin-labeled MAbs to lineage-specific
antigens (B220, CD19, CD3, NK1.1, Ter-119, Gr-1, and Thy-1), and then sorted using a FACS Vantage
cell sorter (Becton Dickinson). Sorted cells consisted routinely of >98% CD11c+ Linneg cells. In some
experiments, the CD11cneg fraction was also sorted and collected separately. Sorted DC were pulsed
with peptide and used as described above.
T cell isolation and T cell/DC co-cultures. Naïve CD8+ P14xT-GFP and OT-IxRag-/- T cells were
purified from splenocytes by RBC lysis in ‘ACK’ lysis buffer followed by negative selection. Briefly,
cells were incubated with 10 g/ml MAbs to B220, I-A/I-E (2G9) and 5 g/ml MAbs to CD4
(H129.19), CD19, Pan-NK, Ter-119, and Gr-1 followed by goat anti-rat IgG microbeads as described
above. T cells (90-95% CD8+/L-selectinhi/CD44lo/GFPhi) were co-cultured with 1 x 106 peptide-pulsed
DC from different lymphoid organs (T:DC ratio was 1:1 or 2:1) in 3 ml complete IMDM using 12-well
tissue-culture treated plates (Falcon). After 3 days, 2 ml complete IMDM plus a single dose of 10 ng/ml
rIL-2 was added to maintain viability, and 2 ml complete IMDM (without rIL-2) were replaced daily
thereafter. Cells were used after 6-7 days in culture. Between days 6 to 8 of culture  expression on
CD8+ effector T cells remained constant. However, cell viability decreased significantly from day 7.
Therefore, for most experiments T cells were harvested at day 6 of culture.
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For some experiments, naïve P14xT-GFP cells were stimulated with and without addition of FACS
sorted CD11c+ or CD11cneg fractions. For this, 12-well plates were coated with 10 g/ml anti-CD3
(145-2C11) and anti-CD28 (37.51) in PBS for 2 hours at 37oC, washed three times, and 1-2 x 106
P14xT-GFP cells were added alone or together with sorted CD11c+ or CD11cneg cells (ratio of T
cells:sorted cells was 1:1 or 2:1). After 3 days in culture, the cells were transferred to wells without
plate bound antibodies, supplemented with a single dose of 10 ng/ml rIL-2 and maintained in complete
IMDM as described above. Of note, we added rIL-2 only once on day 3 because we noticed that more
frequent supplementation or higher doses of this cytokine suppressed the DC induced upregulation of
 on effector cells.
Flow cytometry. All labeling procedures were performed at 4oC in DMEM + 1% FBS + 20 mM Hepes.
DC or T cells were stained with fluorescent primary MAbs and appropriate isotype controls in the
presence of anti-CD16/CD32 using standard procedures. DC and T cells were analyzed after gating on
viable CD11c+ or CD8+ cells, respectively. In some experiments, P14xT-GFP naïve and effector T cells
were labeled with specific gp33-41H-2Db or control np396-404H-2Db MHC-I tetramers (Beckman Coulter).
MAbs and MHC-I tetramers were used at previously determined appropriate dilutions and samples were
analyzed immediately on a FACScan flow cytometer (Becton Dickinson). For  heterodimer
staining (using MAb DATK32), data in fig. 1c were presented as mean  SEM and analyzed using the
Kruskal-Wallis test with Dunn’s post-test.
Chemotaxis assays. 2 x 105 T cells were loaded in the upper well of Transwell filters with 5 µm pore
size (Costar, Corning) with or without chemokines (R &D Systems) in the lower well. After 90 min at
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37oC, cells in the bottom well were collected and counted by flow cytometry. A known concentration of
fluorescent microbeads (Sigma) was added to each sample to normalize cell counts. Chemotactic
indices were calculated as the number of migrated cells in wells containing chemokine, divided by the
number of migrated cells in wells containing medium alone. The percentage of migrated relative to the
number of cells in the input was also determined.
Chemotaxis data are presented as mean  SEM and were analyzed using one-way ANOVA with
Bonferroni correction. For assays using TECK, n = 22 experiments with CD8PP-DC and CD8PLN-DC, 6
with CD8Sp-DC, and 3 with CD8Naïve . Experiments with other chemokines were performed using
concentrations that induced maximal chemotaxis, i.e. 10 nM for RANTES/CCL5 (n = 9), SDF/CXCL12
(n = 13 with CD8PP-DC and CD8PLN-DC, n = 3 with CD8Sp-DC), SLC/CCL21 (n = 9 with CD8PP-DC and
CD8PLN-DC, n = 4 with CD8Sp-DC), MCP-1/CCL2 (n = 2), and 100 nM for TARC/CCL17 (n = 4).
Intracellular cytokine production. Effector T cells were restimulated with PMA plus ionomycin in the
presence of brefeldin A, and analyzed by flow cytometry for intracellular staining of IL-2 (JES6-5H4
clone) and IFN- (XMG1.2 clone) using a commercial kit (Pharmingen).
Cytotoxicity assay. Effector T cells were harvested from cocultures and tested for their specific
cytolytic activity of EL-4 (H-2b) target cells with or without pulsing with 10 g/ml gp33-41 peptide using
a standard 6 hour chromium release assay 4. Specific cytotoxicity was defined as: (test release –
spontaneous release) / (maximum release – spontaneous release) x 100. Background cytotoxicity of
unpulsed EL-4 cells (always <5%) was subtracted.
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Real-Time rt-PCR. Total RNA was isolated from naïve and effector CD8+ P14xT-GFP cells and
digested with RNAse-free DNAse (Promega). Single stranded cDNA was synthesized using random
hexamers and RT-easy Reverse Transcription Kit (Maxim Biotech). Quantitative detection of CCR9
cDNA was performed using an iCycler (Bio-Rad). cDNAs corresponding to equivalents amounts of
starting RNA was amplified using the SYBR Green DNA PCR Core Kit (Applied Biosystems) with the
following primers: CCR9 forward 5’-AGGTTAGTCAGCCAATGTACAGC-3’ and reverse, 5’ATCCTTTCCTAGTTTGTGCTTGC-3’; GAPDH forward 5’-CAACTTTGTCAAGCTCATTTCC-3’
and reverse 5’-GGTCCAGGGTTTCTTACTCC-3’. CCR9 levels were expressed as the CCR9/GAPDH
ratio for each sample. The specificity of PCR amplicons was confirmed by gel electrophoresis.
Contamination by genomic DNA was excluded by parallel analysis of samples without reverse
transcriptase. Data are presented as mean  SEM and analyzed using one-way ANOVA with Bonferroni
correction. n = 3 independent determinations for effector populations and 2 for naïve CD8+ P14xT-GFP
cells.
Homing assay. For comparison of effector T cells with naïve T cells, CD8PLN-DC and CD8PP-DC were
labeled for 20 min at 37˚C with tetra-methylrhodamine isothiocyanate (TRITC, Molecular Probes). 2 x
107 TRITC+ cells were mixed with an equivalent number of splenocytes from T-GFP mice as described
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. Mixtures were injected via the tail vein into non-transgenic recipient mice. An aliquot was saved to
assess the input ratio (IR = [TRITC+]input /[GFP+]input). After 4 hours, recipient mice were sacrificed.
PP, LN, spleen, liver, lungs, and small intestine were harvested and TRITC+/GFP+ ratios in blood and
single-cell suspensions of each tissue were determined by flow cytometry. The homing index (HI) was
calculated as: HI = [TRITC+]tissue/[GFP+]tissue : IR, where IR = [TRITC+]input/[GFP+]input. To compare the
migration of effector cells, P14xT-GFP cells were cocultured for 6-7 days with antigen-pulsed PP DC or
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PLN DC and T cells from one population were labeled with TRITC (Molecular Probes) while the other
population was stained with calcein AM or CFSE (Molecular Probes), green fluorophores. 1.5-2 x 107
cells from each population were mixed and injected into recipient mice. In two experiments, mice were
injected with 100 g IgG2b or anti-TECK (R&D Systems, clone 89818) 4 hours prior to and during
CTL injection. Recipients were sacrificed after 18 hours and single cell suspensions were generated
from multiple recipient tissues as described 5. Isolation of homed cells from the LP and IEL of the small
intestine was performed as described 6. The HI was calculated as: [CD8PP-DC]tissue/[CD8PLN-DC]tissue :
[CD8PP-DC]input/[CD8PLN-DC]input. Data are presented as mean  SEM. Homing indices were tested vs. HI
= 1 using a one-sample Wilcoxon-signed rank test. n = 12 experiments for blood, spleen, PLN, MLN,
PP, IEL/LP, and 3 for appendix, colon, liver and lung.
References
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Manjunath, N. et al. A transgenic mouse model to analyze CD8(+) effector T cell differentiation
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