Human CD28+/- tumor infiltrating lymphocytes gene expression

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. 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.).