Supporting InformationSummers deLuca et al. 10.1073/pnas.1014188108SI Materials and MethodsMice. Wild-type C57BL/6 mice were purchased from CharlesRiver. C57BL/6 OTII mice were purchased from Charles Riverand were bred in-house. Lymphotoxin (LT)β−/− mice purchasedfrom B&K Universal were crossed with OTII mice. bm1 micewere purchased from The Jackson Laboratory and were bred in-house. LTβ-receptor (LTβR)−/− mice were a kind gift fromRodney Newberry (Washington University School of Medicine,St. Louis, MO). All animals were housed under specific pathogen-free conditions and all experiments were performed according toanimal-use protocols approved by the University of Toronto.

Bone Marrow Chimeras. Bone marrow (BM) was collected fromfemurs and tibia of WT C57BL/6 (CD45.1) mice, LTβR−/−

CD45.1 mice, or CD11c-DTR/GFP CD45.2 mice and filteredthrough a 70-μm nylon mesh (BD Falcon), and red blood cellswere lysed using RBC lysing buffer (Sigma-Aldrich). For singlechimeras, 2–4 × 106 BM cells were then injected i.v. into C57BL/6CD45.2 mice that had been lethally irradiated (2 × 550 rad).Recipient mice were left for 8–10 wk to reconstitute, and weregiven water supplemented with 2 mg/mL neomycin sulfate (Bio-Shop) for the first 2 wk. In some cases, BM cells fromWTCD45.2and LTβR−/− CD45.1 donors were mixed 1:1 before transfer intoLTβR+/− CD45.1/2 irradiated recipients, or BM cells fromCD11c-DTR and WT CD45.1 or LTβR−/− CD45.1 were mixed1:1 before transfer into C57BL/6 CD45.2 irradiated mice.

Dendritic Cell Depletion of CD11c-DTR Chimeras. Diphtheria toxin(DT) was obtained from Sigma and reconstituted per the man-ufacturer’s instructions. Six hours before immunization andagain 24 h later, chimeric mice were given DT at 4 ng/g i.p.,diluted to 0.5 μg/mL in sterile PBS. Depletion of CD11c+

(EGFP+) cells was measured in the blood, spleen, and lymphnode (LN) at 24 h post–DT treatment by measuring specificdepletion of EGFP+ dendritic cells (DCs) by flow cytometry(data not shown).

Flow Cytometry. Antibodies against CD4, CD8, CD11c, CD25,CD45.1, CD45.2, CD80, CD86, CD40, MHCII, CD11b, IFNγ,Thy1.1 (FITC,PE-Cy5), Thy12, aswell as streptavidin-fluorochromeconjugates were purchased from eBioscience. Antibodies againstCD69, Thy1.1 (PerCP-Cy5.5), Vα2, and Vβ5.1 were obtained fromBD Biosciences. Antibody against PDCA-1 was purchased fromMiltenyi.All surface stainswereperformed inPBS/2%FBS (Gibco)/

0.05%sodiumazide (EMScience,Merck). Intracellular stainingwasperformed using a BDBiosciences Cytofix/CytopermKit. DyeCycleViolet was obtained from Invitrogen, and was used according to themanufacturer’s instructions. All stained samples were acquired ona FACSCalibur, Canto, or LSRII (BD Biosciences) as appropriate.For the generation of BMDCs, femurs and tibia were removed

from C57BL/6 (LTβR+/+ and LTβR−/−) mice and bone marrowcells were harvested by flushing with PBS. The BMDC cultureswere prepared at a density of 2 × 106 cells/mL in RPMI medium1640 (Sigma-Aldrich) supplemented with 10% FBS (Gibco; in-activated for 60 min at 56 °C), 0.05 mM β-mercaptoethanol, 100U/mL penicillin, 100 μg/mL streptomycin, 40 U/mL murine re-combinant GM-CSF (Peprotech). Fresh medium was added ev-ery 3 d until day 6, and every 2 d thereafter. Nonadherent cellswere harvested on day 10 for analysis.

B3Z Assay for Antigen Presentation. C57BL/6 mice received anadoptive transfer (A/T) of 1 × 106 OTII CD4+ T cells and weretreated i.p. with 100 μg of either LTβR-Ig fusion protein orhuman IgG control. The next day, the mice were immunized i.p.with 25 × 106 OVA-loaded bm1 splenocytes. The mice werekilled 18 h after immunization, and the draining LNs were har-vested and mashed with glass slides. Tissue was digested as DCswere purified as outlined above and cocultured with B3Z re-sponder T cells at the ratio indicated overnight. The cells wereharvested and lysed in 0.1 M K2HPO4, 0.2% Triton X. Chloro-phenol red-β-D-galactopyranoside (CPRG) was added for col-orimetric detection of LacZ activity, and read at 595 nm using anELISA plate reader. Similar experiments were performed usingWT→WT and LTβR−/−→WT BM chimeric mice.

Primer Sequences. Oligonucleotide primers for quantitative PCRanalysis were as follows: for IFNα4 (forward) 5′-GCC ATC CTTGTG CTA AGA G-3′; (reverse) 5′-TCA AGA GGA GGT TCCTGC ATC AC-3′; for IFNα5 (forward) 5′-ACA GGT CGGGGT GCA GGA ATC T-3′; (reverse) 5′-CAC TCC TCC TTGCTC AAT CTT-3′; for IFNp (forward) 5′-TGC GTT CCT GCTGTG CTT CT-3′; (reverse) 5′-TTG GAT GGC AAA GGCAGT GT-3′; for hydroxymethylbilane synthase (HMBS) (for-ward) 5′-TCC AAG AGC CCA GCT A-3′; (reverse) 5′-ATTAAG CTG CCG TGC AAC A-3′. PCR was activated by heatingsamples at 95 °C for 10 min, and cycled at 95 °C for 15 s, 61 °Cfor 60 s for 35 cycles.

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A B

Fig. S1. Representative FACS plots of OTI T-cell expansion/IFNγ production following LTβR-Ig treatment. WT bm1xB6 F1 were given an A/T of responderCD45.1 or Thy1.1 OTI T cells, treated with either control huIgG or LTβR-Ig, and immunized the next day with OVA-loaded bm1 cells. At multiple days post-immunization, OTI expansion (A) and IFNγ production (B) were measured in the spleen. Data are representative of three independent experiments (n = 3–6 perexperiment). Black empty histogram, huIgG-treated; gray empty histogram, LTβR-Ig–treated; gray filled histogram, unimmunized.

Fig. S2. Evaluation of DC phenotype/function in LTβR-Ig treated mice. (A) Mice were treated on day 0 with control huIgG or LTβR-Ig. In some cases, mice wereimmunized with bm1-OVA on day 1 and then splenic DCs were harvested 18 h later and coincubated with the B3Z responder T cells overnight to evaluate theloading of SIINFEKL peptide on MHCI (SIINFEKL:MHCI). SIINFEKL:MHCI presentation was measured by colorimetric detection of β-galactosidase activity usingCPRG substrate. x axis is the ratio of DC:B3Z. These experiments were performed two times with similar results, and results shown represent the average ofthree mice per group. *P < 0.05, ***P < 0.001. NS, nonsignificant. (B and C) Mice were treated on day 0 with control huIgG or LTβR-Ig and splenic DC wereenumerated as a % of total splenocytes (B) or as total DCs per spleen (C) following collagenase D and DNase I digestion. DCs were further analyzed withrespect to the distribution of DC subsets and expression of surface markers, with a representative example shown (D). No statistically significant differenceswere observed between huIgG- or LTβR-Ig–treated groups. The experiment was performed using three mice per group.

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Fig. S3. Evaluation of DC phenotype/function in LTβR−/− chimeric mice. (A) WT→WT or LTβR−/−→WT chimeric mice were analyzed as in Fig. S2A. x axis is theratio of DC:B3Z. This experiment was performed two times with similar results, and results shown represent the average of three mice per group. **P < 0.001.(B and C) Splenic DCs from WT→WT or LTβR−/−→WT chimeric mice were enumerated as a % of total splenocytes (B) or as total DCs per spleen (C). DCs werefurther analyzed as in Fig. S2D (D). No statistically significant differences were observed between WT→WT and LTβR−/−→WT chimeric mice with the exceptionthat the frequency of CD4+CD11b+ DCs was reduced in LTβR−/−→WT chimeric mice. The experiment was performed two times using three mice per group foreach experiment.

Fig. S4. Expression of LTβR on DCs is required for optimal numbers of OTI at the peak of the immune response in vivo. Mixed chimeric WT + CD11c-DTR→WTor LTβR−/− + CD11c-DTR→WT mice were given an A/T of responder CD45.1 OTI T cells, treated with diphtheria toxin, and immunized with OVA-loaded bm1cells, and OTI expansion was evaluated 3 d postimmunization. Number of OTI per spleen was enumerated for each mouse. *P < 0.05.

Fig. S5. Expression of LTαβ on antigen -specific CD4+ T cells is required for optimal expansion of OTI in vivo and in vitro. WT B6 mice were given an A/T ofeither WT or LTβ−/− helper OTII T cells along with responder CD45.1 OTI T cells, and were immunized the next day with OVA-loaded bm1 cells. At 3 d post-immunization, OTI expansion (A) and IFNγ production (B) were measured in the spleen. *P < 0.05. Black bars, unimmunized mice; white bars, immunized micethat received WT OTII T cells; gray bars, immunized mice that received LTβ−/− OTII T cells. The experiment was performed two times with similar results. (C)BMDCs were preincubated with LPS and OVA protein for 18 h, washed, and then plated with OVA-specific carboxyfluorescein succinimidyl ester (CFSE)-labeledOTI T cells and in some cases WT or LTβ−/− OTII T cells. Seventy-two hours later, OTI T cells were assessed for CFSE dilution. This experiment was performed twotimes with similar results.

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Fig. S6. LTβR−/− DCs display altered CD86 up-regulation. WT→WT and LTβR−/−→WT chimeric mice were given an A/T of helper WT OTII T cells and wereimmunized 1 d later with OVA protein and LPS. At 18 h (A) and 36 h (B), draining lymph nodes (dLN) were collected, digested, and stained to measure CD86expression on CD11c+ DCs by flow cytometry. A reduction in CD86 expression on LTβR−/− DCs was observed at 18 h postimmunization, but CD86 expression wasrecovered at 36 h postimmunization. The experiment was performed three times with similar results. *P < 0.05. MFI, mean fluorescence intensity. (C) WTCD45.2 + LTβR−/− CD45.1→LTβR+/− CD45.1/2 mixed bone marrow chimeras were given an A/T of helper WT OTII T cells and were immunized 1 d later with DQ-OVA protein and LPS in one foot. CD86 expression was tracked onWT (filled circles) versus LTβR−/− (empty circles) CD11c+ dLN DCs (black lines) as well as on DQ-OVA+ CD11c+ dLN DCs (gray lines). Note that in the case of mixed BM chimeras, no difference in CD86 was observed, suggesting that WT can rescue CD86expression on LTβR−/− DCs in trans.

A) B)

C)

Fig. S7. LTβR+/− BMDCs express normal levels of costimulation markers before and after LPS stimulation, and are capable of producing IL-12 in vitro. WT (openbars) and LTβR−/− (gray bars) BMDCs were directly stimulated with anti-CD40 and anti-LTβR Abs. At 30 h, DCs were collected and IL-12p40 expression wasevaluated by flow cytometry (A and B), where fold over background indicates the amount of IL-12 expression, expressed as MFI compared with unstimulatedBMDCs. Note that anti-LTβR treatment does not induce IL-12p40 expression. The experiment was repeated two times with similar results. (C) Indicated surfacemarkers on WT and LTβR−/− BMDCs were evaluated before (filled histograms) and after 18-h treatment with LPS (empty histograms). *P < 0.05.

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Fig. S8. Type I IFN production induced by LPS + OVA is attenuated in WT BMDCs by LTβR-Ig treatment. WT BMDCs were coincubated with OVA-specific CD4+ Tcells (OTII) and LPS and OVA323–339. In some cases, these cocultures were also supplemented with LTβR-Ig to block LTβR signaling in DCs. The expression of IFNβ(A) and IFNα5 (B) was measured by real-time RT-PCR. This experiment was performed two times with similar results. *P < 0.05.

Fig. S9. Substitution of LPS/OVA with bm1-OVA in BMDC/OTI cocultures promotes OTI division which is compromised in the presence of LTβR−/− BMDCs. WT orLTβR−/− BMDCs were plated with bm1-OVA protein and CFSE-labeled OVA-specific CD8+ T cells (OTI) with or without OTII CD4+ T cells. Five days later, OTI T cellswere gated based on CD45.1 expression and/or CD8 expression, and then assessed for CFSE dilution and entry into the cell cycle using DyeCycle Violet. Notethat the extent of proliferation is less than what is observed in Fig. 5, and the proliferation is observed on day 5 rather than day 3. For these reasons, LPS/OVAwas judged to be more amenable for in vitro BMDC evaluation. This experiment was performed two times with similar results.

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