Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for a
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BioMed Central
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Methodology
Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy Udai S Kammula* and Oscar K Serrano Address: Surgery Branch, National Cancer Institute, Bethesda, MD, USA Email: Udai S Kammula* - [email protected]; Oscar K Serrano - [email protected] * Corresponding author
Published: 20 October 2008 Journal of Translational Medicine 2008, 6:60
doi:10.1186/1479-5876-6-60
Received: 25 August 2008 Accepted: 20 October 2008
This article is available from: http://www.translational-medicine.com/content/6/1/60 © 2008 Kammula and Serrano; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Background: The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods: We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay) as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results: In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells). Conclusion: This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.
Background Adoptive immunotherapy with autologous tumor infiltrating lymphocytes (TIL) in conjunction with a lymphodepleting conditioning regimen can mediate significant tumor regression in ~50% of patients with refractory metastatic melanoma [1,2]. However, not all patients with melanoma are eligible for this type of
immunotherapy either because resectable tumor is not available, the lymphocytes from the specimen do not expand suffici
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