Human CD28+/- tumor infiltrating lymphocytes gene expression. Homo sapiens

This analysis reveals the different gene expression profiles between human melanoma tumor infiltrating lymphocytes subsets CD8+CD28+ and CD8+CD28-. Adoptive cell therapy (ACT) is an effective approach that removes tumor-infiltrating lymphocytes (TILs) from this suppressive tumor microenvironment and expands and activates CD8+ and CD4+ T cells in vitro, differentiating them into potent anti-tumor effector cells that can be re-introduced back into patients. One of the key problems that may limit tumor regression and long-term durable clinical responses in ACT is the persistence of TILs following infusion. Our study has indicated that ex vivo expanded TILs to be hypo-responsive to peptide re-stimulation, manifested in slow entry into cell cycle and increased apoptosis. Phenotypic and functional analysis using a number of different T-cell differentiation markers found that CD28 was markedly down-modulated in post-REP cells, while CD27 and CD57 levels showed no statistically-relevant changes in expression. Here on a global gene expression level, microarray gene chip analysis of sorted CD8+CD28+ and CD8+CD28- post-REP TILs revealed a number of key differences in their gene expression profiles; notably, an increase in KIR family member expression, loss of p53-binding proteins, and lower CD127/IL-7Ra gene expression found in the CD28- population. On top of advancing T cell phenotype, reactivation and memory, this set of data may also provide insight for ACT clinical protocol optimization to improve TIL response in vivo. Overall design: After isolation from tumor fragment and ex vivo expansion with IL-2, human tumor infiltrating lymphocytes were sorted by FACS into two subsets as described, followed by an immediate microarray analysis. Specifically: The tumor samples were cut into 3-5 mm2 pieces and cultured in 2 ml of TIL culture medium (TIL-CM) consisting of RPMI 1640, 10% human AB serum, 1 mM Glutamine, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol, 1X Penicillin-Streptomcyin (Pen-Strep), and 20 mg/ml Gentmicin (Invitrogen, Carlsbad, CA) in 24-well plates. IL-2, at 6,000 U/ml was added to all wells. The dividing TIL lines were fed with fresh TIL-CM containing 3,000 U/ml IL-2 (Proleukin® from Novartis) and sub-cultured 1:1 in TIL-CM with 6,000 U/ml IL-2 to maintain viable cell density between 0.5-2 x 10e6/ml. These TILs were designated as “pre-REP” and were subjected to continued expansion using the “rapid expansion protocol” (REP) originally designed by Riddell and Greenberg. Briefly, 1.3 x 10e5 pre-REP TILs in TIL-CM were added to upright T-25 flasks containing 30 ng/ml OKT3 and 26 x 10e6 allogeneic irradiated (5,000 cGy) PBMC feeder cells obtained from 4 pooled normal donor PBMC (obtained from the Gulf Coast Regional Blood Bank, Houston, TX) in 20 ml of TIL-CM. On day 2, 10 ml of AIMV (Invitrogen) was added together with 6,000 U/ml IL-2 (final concentration). The TILs were expanded for another 12 days and diluted as needed with AIMV and IL-2 to keep the viable cell density between 1-4 x 106/ml. Using this methodology, routinely 50-100 x 10e6 “post-REP” TIL were isolated from each starting T-25 flask culture. Post-REP TILs were freshly harvested from the flasks and sorted after anti-CD4 and anti-CD28 staining into CD4-, CD28+ and CD4-, CD28- two subsets. After a 3 h rest period to shed off staining antibodies post sorting, TILs were processed with Qiagen RNeasy kit to obtain RNA for microarray experiment. 1 mg of RNA of each sample was subjected to reverse transcription and probe blotting using an Illumina kit, where human Ref6 chip was used (Illumina, Inc.).