Siinfekl peptide

Murine primary peritoneal macrophages or BMDMs were conditioned with 10% B16-S1pr1 TCM for 24 h and incubated with 2 μg/ml SIINFEKL peptide (InvivoGen) for 1.5 h at 37 °C. Cells were stained with an antibody specific for H-2K^b-SIINFEKL complex or an isotype control and analyzed using a flow cytometer.

OVA-expressing target cells (either splenocytes pulsed with 5 μg/mL SIINFEKL peptide [InvivoGen], B16-OVA cells, or EG7 cells) were labeled with CFSE and mixed at a 1:1 ratio with CTO-labeled non-antigen-expressing cells (i.e., non-pulsed splenocytes, B16 cells, or EL4 cells, respectively) and incubated in triplicate in the absence of effector T cells, or in the presence of a 15-fold excess of effector WT OT-I or Cxcr3^−/− OT-I T cells. After 24 h, live cells were analyzed by flow cytometry. For in vivo cytotoxicity assays, OVA-pulsed and non-pulsed splenocytes were mixed at a 1:1 ratio (~4–5 ×10⁶ of each population/mouse) and transferred i.v. into naïve mice in which 3 × 10⁷ WT or Cxcr3^−/− effector OT-I T cells had been injected 3 h previously. 24 h later, spleens were harvested, lysed, and live cells were analyzed by flow cytometry. The proportion of fluorescently-labeled cells was plotted as CFSE⁺ and CFSE⁻ cells on histograms, and the % specific cytotoxicity was determined by the formula [1-(% CFSE⁻ cells/% CFSE⁺ cells)] × 100%.

For activation of polyclonal CD8⁺ T cells, T cells were isolated from the spleen and inguinal lymph nodes of C57BL/6 mice (Jackson 000664) using the Dynabeads Untouched CD8⁺ T cell kit. T cells were activated with plate-bound anti-CD3 (2C11, 3 μg/mL, 3:1000 dilution) and anti-CD28 (19.51, 1 μg/mL, 1:500 dilution) for 48 h in RPMI-1640 media containing 10% FBS, 2 mM L-glutamine, 50 μM β-ME, and 10 ng/mL IL-2 (Peprotech). For activation of OT-I transgenic CD8⁺ T cells, single cell suspensions were generated from spleens and inguinal lymph nodes of OT-I mice (Jackson 003831). Cells were cultured at 10 × 10⁶ cells per mL in the presence of 1 μM SIINFEKL peptide (Invivogen) + for 48 h in RPMI-1640 media containing 10% FBS, 2 mM L-glutamine, 50 μM β-ME, and 10 ng/mL IL-2 (Peprotech).

Peptides containing the mouse MHC-I (H-2K^b)-restricted ovalbumin (OVA) epitope OVA_257–264 (SIINFEKL) modified with a cationic oligolysine (K_n) tail (K₁₀OVA: K₁₀QLESIINFEKL and K₅OVA: K₅QLESIINFEKL) were synthesized and HPLC purified by EZBiolab (Carmel, IN). K₁₀OVA labeled with TAMRA at the N-terminus (TAMRA-K₁₀OVA) was purchased from Biomatik (Wilmington, DE). The purity of all peptides was above 95% as certified by HPLC and MS analyses. SIINFEKL peptide was purchased from Invivogen.

Bone marrow cells (BMCs) were flushed from the tibias and fibulas of C57BL/6 mice using sterile PBS. Red blood cells were lysed (1 minute at RT) using Red Cell Lysis Buffer (Thermo; #A1049201) and washed with R10 medium and resuspended at 2×10⁷ cells/mL. During washing, 10 mL R10 medium supplemented with 10ng/mL GM-CSF (BioLegend; 576306) was added to 10 cm Petri dishes. Once BMCs were resuspended, 5×10⁶ cells were added per plate (dropwise into the center of the dish), incubated at RT for 15 minutes to allow to settle, then incubated at 37°C 5% CO₂ for 3 days. On day 3, 10 mL of GM-CSF supplemented pre-warmed R10 medium was gently added followed by culture for another 3 days. On day 6, 90% of the medium was aspirated, replaced with R10 medium supplemented with 10 ng/mL GM-CSF, 1 μg/mL LPS (Escherichia coli O111:B4, Sigma Aldrich; #LPS25), and 1nM SIINFEKL peptide (Invivogen; #vac-sin), and BM-DCs were then incubated overnight. On day 7, BM-DCs were gently lifted using a cell scraper, washed (300 × g, 5 mins), and resuspended at a final density of 1×10⁵ cells/mL in R10 medium.