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. 2015 Apr 28:6:196.
doi: 10.3389/fimmu.2015.00196. eCollection 2015.

Single-Cell Characterization of in vitro Migration and Interaction Dynamics of T Cells Expanded with IL-2 and IL-7

Johanna Tauriainen  1 , Karin Gustafsson  2 , Mårten Göthlin  2 , Jens Gertow  3 , Marcus Buggert  1 , Thomas W Frisk  2 , Annika C Karlsson  1 , Michael Uhlin  3 , Björn Önfelt  4
Affiliations

Single-Cell Characterization of in vitro Migration and Interaction Dynamics of T Cells Expanded with IL-2 and IL-7

Johanna Tauriainen et al. Front Immunol. .

Abstract

T cells are pivotal in the immune defense against cancers and infectious agents. To mount an effector response against cancer cells, T cells need to migrate to the cancer-site, engage in contacts with cancer cells, and perform their effector functions. Adoptive T cell therapy is an effective strategy as treatment of complications such as relapse or opportunistic infections after hematopoietic stem cell transplantations. This requires a sufficient amount of cells that are able to expand and respond to tumor or viral antigens. The cytokines interleukin (IL)-2 and IL-7 drive T cell differentiation, proliferation, and survival and are commonly used to expand T cells ex vivo. Here, we have used microchip-based live-cell imaging to follow the migration of individual T cells, their interactions with allogeneic monocytes, cell division, and apoptosis for extended periods of time; something that cannot be achieved by commonly used methods. Our data indicate that cells grown in IL-7 + IL-2 had similar migration and contact dynamics as cells grown in IL-2 alone. However, the addition of IL-7 decreased cell death creating a more viable cell population, which should be beneficial when preparing cells for immunotherapy.

Keywords: IL-2; IL-7; T cell; fluorescence; live-cell imaging; microchip; microscopy; single-cell analysis.

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Figures

Figure 1
Figure 1
Effect of IL-7 on T cell expansion and phenotype. T cells were positively selected and cultured for 7 days with anti-CD3- and anti-CD28-coated beads and IL-2 + IL-7 or IL-2 alone, after which the cells were counted and phenotyped by flow cytometry and the expressions of CD4, CD8, CCR7, CD45RO, γδ-TCR, CD25, and CD39 were compared between the two culture conditions. (A) Relative T cell expansion displayed as ratio of fold increase in IL-2 + IL-7 over IL-2. (B) Kinetics of fold expansion for one representative donor. This donor gave median fold expansion after 7 days for both culture conditions. (C) CD4/CD8 ratio. (D) Distribution of memory subsets. T cell maturation profile from least mature to most mature was defined as: CCR7+CD45RO− (naïve, red); CCR7+CD45RO+ (early-differentiated, blue); CCR7−CD45RO+ (intermediate-differentiated, yellow); CCR7−CD45RO− (late-differentiated, orange). (E) Percentage of CD3+CD4−CD8−T cells expressing γδ-TCR. (F) Percentage of regulatory T cells, defined as CD3+CD4+CD127−CD25+CD39+. Flow cytometry data are based on data from 9 individual donors, and cell expansion data is based on 12 individual experiments. Lines connect cultures from the same donor, horizontal lines in (A) depict the median and IQR. Mann–Whitney tests were performed to evaluate significance between the groups.
Figure 2
Figure 2
Long-term live-cell imaging in microwells for characterization of migration and contact dynamics. (A) Schematic picture of the device consisting of a metal holder (1), multi-well silicon-glass microchip (2), PDMS gasket (3), and lid (4). The lid was held tightly against the metal holder by magnets. The PDMS gasket was cut to create two separate basins (one per condition), each containing approximately 30 microwells. (B) Scanning electron microscopy image showing a subsection of the microchip. The dimension of the microwell bottom was 450 × 450 μm2 and the well depth was 300 μm. (C) Fluorescence image of a microwell containing T cells (red) and target cells (green). (D) Trajectories from T cells (n = 23) followed in a representative time-lapse sequence.
Figure 3
Figure 3
Addition of IL-7 does not influence T cell migration and target cell interaction dynamics. (A) Box plot of T cell migration speed for the two conditions. The mean speed of T cells cultured in IL-2 (red, n = 219, median 2.8 μm/min), and T cells cultured in IL-2 + IL-7 (blue, n = 175, median 2.8 μm/min). (B) The fraction of time (%) spent in contact with target cells shown for cells cultured in IL-2 (red, n = 219) or IL-2 + IL-7 (blue, n = 175) during the time interval they were followed. (C) Boxplot of the duration of all individual contacts scored between T and target cell during the 7-h assay for the two conditions (n = 152 for IL-2 and n = 125 for IL-2 + IL-7). Median times per contact were 8 min for both conditions. (D) Histogram showing the number of contacts made with target cells by individual T cells (%) during the 7-h assay for cells cultured in IL-2 (red bars, n = 219 cells) or in IL-2 + IL-7 (blue bars, n = 175 cells). Mann–Whitney tests were performed to compare mean speed, fraction of time in contact, time spent in each individual contact, and number of contacts made by each T cell between the two culture conditions.
Figure 4
Figure 4
T cell–target cell interactions were brief or more stable, sometimes leading to target cell death. (A) Time-lapse sequence showing a T cell forming a brief contact with a target cell. The panels show transmitted light (top), fluorescence showing T cells in red and target cells in green (middle) and fluorescence overlaid with the T cell trajectory (white line, bottom). A T cell (marked by arrowheads) migrated toward a target cell (frame 1), formed a contact with the target cell (frame 2), continued migrating while attached to the target cells, scanning the target cell surface (frames 3 and 4), and finally detached without forming a stable conjugate or killing the target cell (frame 5). (B) Time-lapse sequence showing T cell-mediated target cell death. Panels as in (A). A T cell (marked by arrowheads) migrated with an elongated shape (frame 1), made a contact with a target cell within a cluster of three cells (frame 2), rounded up, and formed a conjugate (frame 3), which was followed by target cell death seen as a decreased green fluorescence intensity and a cloud of cellular debris in the transmitted light channel (frames 4 and 5). Indicated times are hours:minutes and scale bar represent 20 μm. Images have been resampled and brightness and contrast altered to improve visibility.
Figure 5
Figure 5
Addition of IL-7 had no effect on rate of T cell mitosis but decreased cell death. (A) Percentage of T cells undergoing mitosis during the 7-h assay for the two conditions. (B) Time-lapse imaging data of cell division shown in transmission (top) and fluorescence (bottom). A T cell (red fluorescence) migrated with a typically elongated, migratory shape (frame 1) and stopped, enlarged, and rounded up (frame 2), this interphase was followed by mitosis where the cells split into two daughter cells (frames 3 and 4) and finally cytokinesis (frame 5), where the new cells were divided. Indicated times are hours:minutes and the scale bar represents 10 μm. (C) Percentage of T cells dying during the 7-h assay for the two conditions (p < 0.005). (D) Time-lapse imaging data of T cell death shown in transmission (top), fluorescence (bottom). A migrating T cell (red fluorescence) stopped and rounded up (frame 1), followed by cell death seen as membrane blebbing in the transmitted light image (top) and decreased red fluorescence intensity (bottom) (frames 2–5). Indicated times are hours:minutes and scale bar represents 10 μm. Chi-squared test was used to compare the rate of cell death and mitosis between the two culture conditions. The data in (A,C) are based on three individual experiments (IL-2 n = 219, IL-2 + IL-7 n = 175). Images have been resampled and brightness and contrast altered to improve visibility.
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References

    1. Cole DJ, Taubenberger JK, Pockaj BA, Yannelli JR, Carter C, Carrasquillo J, et al. Histopathological analysis of metastatic melanoma deposits in patients receiving adoptive immunotherapy with tumor-infiltrating lymphocytes. Cancer Immunol Immunother (1994) 38(5):299–303.10.1007/BF01525507 - DOI - PMC - PubMed
    1. Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, Moen R, et al. Gene transfer into humans – immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N Engl J Med (1990) 323(9):570–8.10.1056/NEJM199008303230904 - DOI - PubMed
    1. Uhlin M, Gertow J, Uzunel M, Okas M, Berglund S, Watz E, et al. Rapid salvage treatment with virus-specific T cells for therapy-resistant disease. Clin Infect Dis (2012) 55(8):1064–73.10.1093/cid/cis625 - DOI - PubMed
    1. Rooney CM, Smith CA, Ng CY, Loftin SK, Sixbey JW, Gan Y, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein–Barr virus-induced lymphoma in allogeneic transplant recipients. Blood (1998) 92(5):1549–55. - PubMed
    1. Porter DL, Levine BL, Bunin N, Stadtmauer EA, Luger SM, Goldstein S, et al. A phase 1 trial of donor lymphocyte infusions expanded and activated ex vivo via CD3/CD28 costimulation. Blood (2006) 107(4):1325–31.10.1182/blood-2005-08-3373 - DOI - PubMed