Expansion and Antitumor Cytotoxicity of T-Cells Are Augmented by Substrate-Bound CCL21 and Intercellular Adhesion Molecule 1

Abstract

Adoptive immunotherapy is based on ex vivo expansion and stimulation of T-cells, followed by their transfer into patients. The need for the ex vivo culturing step provides opportunities for modulating the properties of transferred T-cells, enhancing their antitumor abilities, and increasing their number. Here, we present a synthetic immune niche (SIN) that increases the number and antitumor activity of cytotoxic CD8⁺ T-cells. We first evaluated the effect of various SIN compositions that mimic the physiological microenvironment encountered by T-cells during their activation and expansion in the lymph node. We found that substrates coated with the chemokine CCL21 together with the adhesion molecule intercellular adhesion molecule 1 significantly increase the number of ovalbumin-specific murine CD8⁺ T-cells activated by antigen-loaded dendritic cells or activation microbeads. Notably, cells cultured on these substrates also displayed augmented cytotoxic activity toward ovalbumin-expressing melanoma cells, both in culture and in vivo. This increase in specific cytotoxic activity was associated with a major increase in the cellular levels of the killing-mediator granzyme B. Our results suggest that this SIN may be used for generating T-cells with augmented cytotoxic function, for use in cancer immunotherapy.

Keywords: CCL21; T-cell clusters; T-cell cytotoxicity; T-cell immunity; cancer immunotherapy; intercellular adhesion molecule 1.

Figure 1

Substrate-immobilized CCL21 increases the size of CD8⁺ T-cell clusters, while substrate-immobilized intercellular adhesion molecule 1 (ICAM1) transforms the clusters into substrate-attached monolayers. (A1–D2) Transmitted light microscopy images depicting morphological changes in co-cultures of OT-I CD8⁺ T-cells and ovalbumin-loaded dendritic cells (DCs). Primary T-cells and DCs were isolated from spleen and were immediately co-cultured for 72 h, on either an uncoated substrate (A1,A2), substrate-immobilized CCL21 (B1,B2), substrate-immobilized ICAM1 (C1,C2), or substrate-immobilized CCL21 + ICAM1 (D1,D2). Substrate-immobilized CCL21 (B1,B2) induced larger T-cell clusters compared to the uncoated substrate (A1,A2), while ICAM1 alone (C1,C2), or in combination with CCL21 (D1,D2), induced cell spreading and decreased cluster size. Scale bar in A1, B1, C1, and D1: 200 µm. Black squares (A2–D2) show enlarged regions, with a scale bar of 100 µm.

Figure 2

Substrate-immobilized CCL21 + intercellular adhesion molecule 1 (ICAM1) increase cytotoxic T-cell number and proliferation. (A–D) Representative fluorescence images of T-cells grown on different coated substrates for 72 h (A,B) or 7 days (C,D), following the breakdown of cell clusters, and their spin-down. Cell nuclei are stained blue in all cells, and red only in dead cells. Scale bar: 50 µm. (E–H) Viable cell numbers and percentage of dead cells at 72 h (E,G, respectively) and 7 days (F,H, respectively), quantified using automated image analysis. Data are from one experiment representative of at least three independent experiments with 20 replicates each (see Figure S1A in Supplementary Material). Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure. The number of T-cells seeded per well was 3 × 10³. CCL21 and ICAM1 coatings collectively increase viable cell numbers by up to ninefold, without significantly affecting cell death. (I,J) Histogram and bar graph illustrating the increase in cell proliferation induced by CCL21 + ICAM1, indicated by a decrease in the mean fluorescent intensity of CFSE, compared to cells grown on the uncoated culture (data are representative of three independent experiments with four replicates each). Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure]. Histograms in (I) show CFSE levels: green—non-activated T-cells; black —T-cells activated on uncoated substrates; blue—T-cells activated on CCL21 + ICAM1-coated substrates.

Figure 3

Coating with CCL21 + intercellular adhesion molecule 1 (ICAM1) increases proliferation of T-cells activated with αCD3/αCD28 microbeads, but with a smaller cell yield, compared to their activation with antigen-loaded dendritic cells (DCs). (A) Bar graph illustrating live cell number, measured using a metabolic cell viability assay of T-cells activated with either antigen-loaded DCs or activation microbeads, with or without CCL21 + ICAM1 substrate coating. Data are representative of at least three independent experiments with five replicates each. Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure. The number of T-cells seeded per well was 30 × 10³. (B–E) Representative images demonstrating the higher cell density in cultures with CCL21 + ICAM1 compared to no coating, activated with either activation beads (B,C) or antigen-loaded DCs (D,E) for 5 days. Scale bars: 50 µm. (F,G) Histogram and bar graph illustrating the increase in cell proliferation induced by CCL21 + ICAM1, indicated by a decrease of 6.5-fold in the mean fluorescent intensity of CFSE, compared to cells cultured on uncoated substrates [data are from one experiment representative of three independent experiments with four replicates each (see Figure S1B in Supplementary Material). Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure]. Histograms in (F) show CFSE levels: green—non-activated T-cells; black—T-cells activated on uncoated substrates; blue—T-cells activated on substrates coated with CCL21 + ICAM1.

Figure 4

Substrate-immobilized CCL21 + intercellular adhesion molecule 1 augment the killing efficiency of cytotoxic T-cells in vitro. CD8⁺ T-cells, pre-cultured for 72 h (A–C) or 7 days (D–F), were subsequently co-cultured with B16-ovalbumin-GFP cells, in a 3:1 ratio, respectively. (A–F) Representative fluorescence microscopy images, stitched so that each displays an entire well in a 384-well plate; live B16 cells (expressing GFP) are seen in white. (G,H) Bar graphs illustrating the number of viable B16-ovalbumin cells, in co-cultures with T-cells that were pre-cultured for 72 h (G) or 7 days (H), as quantified using automated image analysis [data are from one experiment representative of at least three independent experiments with 10 replicates each (see Figure S3A in Supplementary Material). Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure]. Scale bar: 200 µm.

Figure 5

CD8⁺ T-cells pre-cultured on substrate-immobilized CCL21 + intercellular adhesion molecule 1 (ICAM1) kill target cells more rapidly. (A1–C5) Representative overlays of time-lapse phase contrast and florescence imaging. OT-I CD8⁺ T-cells (unstained) were pre-cultured for 7 days prior to co-culturing with target cells, on either an uncoated substrate (A1–A5,B1–B5) or on substrate-immobilized CCL21 + ICAM1 (C1–C5). T-cells were then co-cultured with either B16-GFP cells (A1–A5) or B16-ovalbumin-GFP cells (B1–C5). Live target cells are shown in green. B16-GFP cells, which do not express ovalbumin, were not killed by OT-I T-cells (A1–A5). Substrate-immobilized CCL21 + ICAM1 induced faster killing of B16-ovalbumin-GFP cells (C1–C5), compared to OT-I T-cells pre-cultured on uncoated substrates (B1–B5). Time stamp: hh:mm:ss. Scale bar: 50 µm. (D1–F3) Representative scanning electron micrographs of B16-ovalbumin-GFP cells (large cells spread on the substrate), cultured alone (D1–D3), or co-cultured for 16 h with OT-I T-cells pre-cultured for 7 days on either an uncoated substrate (E1–E3), or on substrate-immobilized CCL21 + ICAM1 (F1–F3). T-cells grown on substrate-immobilized CCL21 + ICAM1 (F1–F3) killed more target cells, as demonstrated by the lower number of remaining B16 cells. Representative target cells are denoted with red stars. Representative T-cell clusters are denoted with blue ellipsoids. Representative isolated T-cells are denoted with yellow triangles. Scale bar: In E1, F1—100 µm; in E2, E3, F2, and F3—10 µm.

Figure 6

Substrate-immobilized CCL21 + ICAM1 increase T-cell expression of Granzyme B and PD-1, while not affecting FasL. (A–C) Bar graph illustrating the mean fluorescence intensity of granzyme B (A), FasL (B), and PD-1 (C) in cytotoxic T-cells following their incubation for 24 and 48 h with B16-ovalbumin target cells. T-cells were pre-cultured for 7 days on either an uncoated substrate, or on substrate-immobilized CCL21 + ICAM1 [data are from one experiment representative of three independent experiments with four replicates each (see Figure S4 in Supplementary Material). Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure].

Figure 7

Substrate-immobilized CCL21 + intercellular adhesion molecule 1 (ICAM1) augment tumor suppression by CD8⁺ T-cells in vivo. (A,B) C57BL/6 mice were injected with 2 × 10⁶ B16 cells expressing ovalbumin and GFP. Seven days later, tumor-bearing mice were split into treatment groups, according to similar tumor average size and distribution, and injected intravenously with 2 × 10⁶ (A) or 4 × 10⁶ activated OT-I T-cells (B). Averages of measured tumor volumes are shown for either untreated mice (gray), or for mice treated with OT-I T-cells pre-cultured for 7 days on an uncoated substrate (blue), or on substrate-immobilized CCL21 + ICAM1 (pink). (C1–D2) Images of histology sections of three representative tumors of mice treated with 2 × 10⁶ OT-I T-cells pre-cultured on uncoated substrates (D1), or on substrate-immobilized CCL21 + ICAM1 (D2); or treated with 4 × 10⁶ OT-I T-cells pre-cultured on uncoated substrates (E1), or on substrate-immobilized CCL21 + ICAM1 coating (E2). Scale bar: 5 mm. T-cells pre-cultured on substrate-immobilized CCL21 + ICAM1 suppressed tumor growth to a significantly greater extent than T-cells cultured on uncoated substrates. N = 10 mice per group. Error bars represent SEM.