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| Status |
Public on Aug 25, 2017 |
| Title |
Activation of human CD8+ T cells in renal cell carcinoma |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
BACKGROUND: Mammalian microRNAs (miR) regulate the expression of genes relevant for the development of adaptive and innate immunity against cancer. Since T cell dysfunction has previously been reported in patients with renal cell carcinoma (RCC; clear cell type), we aimed to analyse these immune cells for genetic and protein differences when compared to normal donor T cells freshly after isolation and 35 days after in vitro stimulation (IVS) with HLA-matched RCC tumor cells.
METHODS: We investigated gene expression profiles of tumor-reactive CD8+ T cells obtained from RCC patient and compared with their HLA-matched healthy sibling donors by microarray approach. In addition, miRNAs analysis was performed in a validation cohort of peripheral blood CD8+ T cells from 25 RCC patients compared to 15 healthy volunteers.
RESULTS: We observed that CD8+ T cells from RCC patients expressed reduced levels of anti-apoptotic and proliferation-associated gene products when compared with normal donor T cells both pre- and post-IVS. In particular, JAK3 and MCL-1 were down-regulated in patient CD8+ T cells versus their normal counterparts, likely due to defective suppressor activity of miR-29b and miR-198 in RCC CD8+ T cells. Indeed, specific inhibition of miR-29b or miR-198 in isolated peripheral blood mononuclear cells (PBMCs) from RCC patients, resulted in the up-regulation of JAK3 and MCL-1 proteins and significant improvement of cell survival in vitro.
CONCLUSIONS: Our results suggest that miR-29b and miR-198 dysregulation in RCC patient CD8+ T cells is associated with dysfunctional immunity and foreshadow the development of miR-targeted therapeutics to correct such T cell defects in vivo.
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| Overall design |
CD8+ T cells were isolated from the PBMCs of autologous and allogeneic normal, healthy donors by negative selection using specific MACS magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) on MiniMACS columns, following the manufacturer's protocol and subsequently assessed for their functional and phenotypic profiles. For MLTC experiments, PBMCs from RCC patients or HLA-matched healthy donors were co-cultured in 24-well plates (Costar, Corning, USA) at 10^6 cells/well with irradiated RCC stimulator cells (10^5 cells/well) in AIM-V medium (Life Technologies, Invitrogen, Italy) supplemented with 10% heat-inactivated pooled human serum (Sigma (medium Mb)). Recombinant human IL-2 was added on day 3 (250 IU/mL; Proleukin, Chiron, and Emeryville, CA). Responder lymphocytes were restimulated weekly with 10^5 irradiated tumor cells in IL-2-containing medium for 2 weeks prior to responder CD8+ T cells isolation. Positively isolated CD8+ T cells were cultured for an additional 2 weeks. On day 0 and day 35 of culture, CD8+ T cells were analyzed for gene and miRNA expression profiles. Responder lymphocytes were restimulated weekly with 10^5 irradiated tumor cells in IL-2-containing medium for 2 weeks prior to responder CD8+ T cells isolation. Positively isolated CD8+ T cells were cultured for an additional 2 weeks. On day 0 and day 35 of culture, CD8+ T cells were analyzed for gene expression profiles. Total RNA was isolated from freshly-isolated and day 35 cultured CD8+ T cells using TRIzol© reagent (Life Technologies), according to the manufacturer's instructions. Total RNA integrity was assessed by electrophoresis using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). Total RNA was processed and hybridized on to the GeneChip Human Genome U133A Array (Affymetrix, Santa Clara, CA), which contains 22,283 gene probe sets, representing approximately 12,000 well-characterized human genes (Affymetrix, Santa Clara, CA).
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| Contributor(s) |
Gigante M, Pontrelli P, Herr W, Gigante M, D’Avenia M, Zaza G, Cavalcanti E, Accetturo M, Lucarelli G, Battaglia M, Storkus WJ, Gesualdo L, Ranieri E |
| Citation(s) |
27063186 |
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| Submission date |
Nov 23, 2006 |
| Last update date |
Aug 10, 2018 |
| Contact name |
Paola Pontrelli |
| E-mail(s) |
[email protected]
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| Phone |
+390805478868
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| Organization name |
University of Bari
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| Department |
Dept. of Precision and Regenerative Medicine and Ionian Area (DIMEPRE-J)
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| Lab |
Nephrology Unit
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| Street address |
Piazza G.Cesare 11
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| City |
Bari |
| ZIP/Postal code |
70124 |
| Country |
Italy |
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| Platforms (1) |
| GPL96 |
[HG-U133A] Affymetrix Human Genome U133A Array |
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| Samples (18)
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| GSM1686405 |
CD8+ T Lymphocytes_TC RCC patient Time 0_replicate 1 |
| GSM1686406 |
CD8+ T Lymphocytes_TC RCC patient Time 0_replicate 2 |
| GSM1686407 |
CD8+ T Lymphocytes_TC RCC patient Time 0_replicate 3 |
| GSM1686408 |
CD8+ T Lymphocytes_TC RCC patient Time 35_replicate 1 |
| GSM1686409 |
CD8+ T Lymphocytes_TC RCC patient Time 35_replicate 2 |
| GSM1686410 |
CD8+ T Lymphocytes_TC RCC patient Time 35_replicate 3 |
| GSM1686411 |
CD8+ T Lymphocytes_TO Healthy donor 2 HLA-matched Time 0_replicate 1 |
| GSM1686412 |
CD8+ T Lymphocytes_TO Healthy donor 2 HLA-matched Time 0_replicate 2 |
| GSM1686413 |
CD8+ T Lymphocytes_TO Healthy donor 2 HLA-matched Time 0_replicate 3 |
| GSM1686414 |
CD8+ T Lymphocytes_TO Healthy donor 2 HLA-matched Time 35_replicate 1 |
| GSM1686415 |
CD8+ T Lymphocytes_TO Healthy donor 2 HLA-matched Time 35_replicate 2 |
| GSM1686416 |
CD8+ T Lymphocytes_TO Healthy donor 2 HLA-matched Time 35_replicate 3 |
| GSM1686417 |
CD8+ T Lymphocytes_IV Healthy donor 3 HLA-matched Time 0_replicate 1 |
| GSM1686418 |
CD8+ T Lymphocytes_IV Healthy donor 3 HLA-matched Time 0_replicate 2 |
| GSM1686419 |
CD8+ T Lymphocytes_IV Healthy donor 3 HLA-matched Time 0_replicate 3 |
| GSM1686420 |
CD8+ T Lymphocytes_IV Healthy donor 3 HLA-matched Time 35_replicate 1 |
| GSM1686421 |
CD8+ T Lymphocytes_IV Healthy donor 3 HLA-matched Time 35_replicate 2 |
| GSM1686422 |
CD8+ T Lymphocytes_IV Healthy donor 3 HLA-matched Time 35_replicate 3 |
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| Relations |
| BioProject |
PRJNA99565 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE6357_RAW.tar |
32.5 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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