Magnetic bead selection

BLT hu-mice were generated as described previously^{25 ,28 (link),37 (link)–39 (link)}. Briefly, human fetal liver and thymus tissues (ca. 1-mm³, Advanced Bioscience Resources, Alameda, CA) were implanted under the kidney capsule of female NSG mice (6- to 8-week-old, Jackson Laboratories, Ellsworth, ME). The mice were bred at The Scripps Research Institute, and each cohort was produced using tissues from a single donor. Magnetic bead selection (Miltenyi Biotec, San Diego, CA) was used to isolate CD34⁺ HSPCs from autologous fetal liver tissue. The purified CD34⁺ cells (Miltenyi Biotec, San Diego, CA) were phenotyped cytometrically^{25 ,28 (link),37 (link)–39 (link)}, and cryopreserved until injection (200,000–350,000 CD34⁺ cells) into mice 3 weeks after Thy/Liv implantation. Human reconstitution in peripheral blood was verified by flow cytometry using previously described methods^{25 ,28 (link),37 (link)–39 (link)}, and the degree of humanization was determined using a gating strategy described elsewhere^{40 (link)}. Mice with an average > 65% of human CD45⁺ cells were selected to ensure successful HIV-1 infection.

AFP-loaded DC (via AdVhAFP transduction or AFP protein pulse) from HLA-A2+ HD or HCC patients were co-cultured with autologous monocyte-depleted PBMC at a 1:10 ratio (DC: PBMC) in RPMI 1640 plus 10 % human AB serum plus pen/strep/L-G In plus 20 IU/ml IL-2 and 5 ng/ml IL-7. Cultures were supplemented with fresh media containing IL-2 and IL-7 every 3–4 days. After 12–13 days, HD CD4+ T cells and HCC patient CD8+ and CD4+ T cells were analyzed for AFP-specific cytokine production, while HD CD8+ T cells were isolated by magnetic bead selection (Miltenyi Biotec) and restimulated with autologous DC (loaded with AFP as in the initial stimulation) for an additional 10d, and then analyzed for AFP-specific cytokine production. For CD8+ T cell intracellular cytokine staining, cells were stimulated in the presence of brefeldin A with T2 cells pre-loaded with one of three immunodominant AFP-derived peptides (AFP_137–145, AFP_158–166, and AFP_325–334; all at 10 μg/ml). Six hours later, T cells were stained for CD8, fixed, permeabilized, and stained for intracellular TNF-α. For CD4+ T cell intracellular cytokine staining, cells were stimulated in the presence of brefeldin A with autologous DC pre-loaded with the AFP peptide pool (at 60 μg/ml). Six hours later, T cells were stained for CD4, fixed, permeabilized, and stained for intracellular IL-2 and TNF-α.

To isolate splenic CD4⁺ RTEs, cells from Rag1-GFP WT and Rag1-GFP CD4-cre Runx1 cKO mice were harvested and first subject to negative selection using a biotin-labeled cocktail of antibodies (B220, CD11b, Ter119, Gr-1, CD19, CD11c and CD8) to eliminate unwanted populations using magnetic bead selection (Miltenyi Biotec) prior to sorting. All sorting was done on a FACSAria (BD Bioscience), and RTEs were sorted by gating on CD4⁺ CD44⁻CD62L⁺ Rag1-GFP⁺. mRNA was later isolated from sorted CD4⁺ RTEs using an RNeasy Mini kit (QIAGEN). cDNA was then generated and amplified with the Ovation PicoSL WTA V2 kit (NuGen). The mRNA expression was measured using TaqMan probes (Applied Biosystems) for ST8Sia1, ST8Sia4, ST8Sia6, ST3Gal1, ST3Gal2 and ST3Gal6. TaqMan probe for 18S rRNA is used as the internal control. Samples were analyzed using an ABI RT-PCR StepOne Plus System (Applied Biosystems), and relative gene expression was calculated via the 2-ΔΔCT method. Data shown is from independent isolations of CD4+ RTEs from 3 Rag1-GFP WT and 3 Rag1-GFP CD4-cre Runx1 cKO mice.

Following cell lines KMS11, H929, RPMI8226, U266, K562 were purchased from ATCC (Manassas, VA, USA). OCI-MY5, OPM2, ARK, RPMI8226/R5 (MM-resistant cell line) were a kind gift from Professor Zhan (Department of Internal Medicine, University of Iowa, Iowa, USA). K562 was maintained in Iscove's Modified Dubecco's Medium (Gibco), supplemented with 10% fetal bovine-serum (Gibco) and 1% penicillin streptomycin–glutamine (Gibco). Other cells were grown in suspension in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine-serum (15% for U266) and 1% penicillin streptomycin–glutamine. All cells were incubated at 37°C in 5% carbon dioxide air atmosphere. CD138⁺ myeloma cells and CD56⁺/CD3⁻ NK cells were isolated from bone marrow and peripheral blood using magnetic bead selection (Miltenyi Biotech, Auburn, CA). Informed consent was obtained from each patient. All selected cells were more than 95% pure.