Abstract
Adoptive T-cell transfer therapy relies upon in vitro expansion of autologous cytotoxic T cells that are capable of tumor recognition. The success of this cell-based therapy depends on the specificity and responsiveness of the T cell clones before transfer. During ex vivo expansion, CD8+ T cells present signs of replicative senescence and loss of function. The transfer of nonresponsive senescent T cells is a major bottleneck for the success of adoptive T-cell transfer therapy. Quantitative methods for assessing cellular age and responsiveness will facilitate the development of appropriate cell expansion and selection protocols. Although several biomarkers of lymphocyte senescence have been identified, these proteins in isolation are not sufficient to determine the age-dependent responsiveness of T cells. We have developed a multivariate model capable of extracting combinations of markers that are the most informative to predict cellular age. To acquire signaling information with high temporal resolution, we designed a microfluidic chip enabling parallel lysis and fixation of stimulated cell samples on-chip. The acquisition of 25 static biomarkers and 48 dynamic signaling measurements at different days in culture, integrating single-cell and population based information, allowed the multivariate regression model to accurately predict CD8+ T-cell age. From surface marker expression and early phosphorylation events following T-cell receptor stimulation, the model successfully predicts days in culture and number of population doublings with R2=0.91 and 0.98, respectively. Furthermore, we found that impairment of early signaling events following T cell receptor stimulation because of long term culture allows prediction of costimulatory molecules CD28 and CD27 expression levels and the number of population divisions in culture from a limited subset of signaling proteins. The multivariate analysis highlights the information content of both averaged biomarker values and heterogeneity metrics for prediction of cellular age within a T cell population.
Highlights
From the ‡Interdisciplinary Program in Bioengineering, Georgia Institute of Technology, USA; §Wallace H
To enable quantification of the “age” of T cells as they expand in culture through combinations of biomarkers, we applied a partial least square regression (PLSR) modeling analysis from data obtained under conditions consistent with in vitro expansion prior to adoptive transfer in patients
Progress has been achieved toward improved T-cell expansion methods in the past few years [53]: new culture media as well as improved stimulation techniques have been developed (54 – 56), and cord blood as been harnessed as a source of nonsenescent lymphocytes for tumor immunotherapy [57]
Summary
Cell Isolation and Expansion—Following institutional review board approval, CD8ϩ T cells were obtained from blood donors using standard isolation procedures. 40 ml of fresh blood was collected in EDTA coated tubes from four healthy donors (21–35 years old) under written informed consent. Peripheral blood mononuclear cells were isolated by density centrifugation using Lymphoprep (VWR), and CD8ϩ T cells further purified using the Dynabeads® UntouchedTM Human CD8 T Cells isolation kit (Invitrogen) (Ͼ92% purity as checked by flow cytometry). The cells were expanded in RPMI 1640 medium with L-glutamine (Sigma- Aldrich) with 10 mM HEPES, 1 mM sodium pyruvate, and 1ϫ modified Eagle’s medium
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