Therapeutic ex vivo T-cell expansion is limited by low rates and T-cell products of limited functionality. Here we describe a system that mimics natural antigen-presenting cells (APCs) and consists of a fluid lipid bilayer supported by mesoporous silica micro-rods. The lipid bilayer presents membrane-bound cues for T-cell receptor stimulation and costimulation, while the micro-rods enable sustained release of soluble paracrine cues. Using anti-CD3, anti-CD28, and interleukin-2, we show that the APC-mimetic scaffolds (APC-ms) promote two- to tenfold greater polyclonal expansion of primary mouse and human T cells compared with commercial expansion beads (Dynabeads). The efficiency of expansion depends on the density of stimulatory cues and the amount of material in the starting culture. Following a single stimulation, APC-ms enables antigen-specific expansion of rare cytotoxic T-cell subpopulations at a greater magnitude than autologous monocyte-derived dendritic cells after 2 weeks. APC-ms support over fivefold greater expansion of restimulated CD19 CAR-T cells than Dynabeads, with similar efficacy in a xenograft lymphoma model.
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This work was supported by the National Institutes of Health (NIH) (1R01EB015498 U01 CA214369) and the Wyss Institute for Biologically Inspired Engineering at Harvard University. D.K.Y.Z. was supported by the Canadian Institutes of Health Research (CIHR-DFSA). S.T.K. was supported by an HHMI ISRF. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. 1541959. CNS is part of Harvard University. We thank the National Institutes of Health (NIH) Tetramer Core Facility for the SIINFEKL/H-2K(b) biotinylated monomer, Alexa Fluor 647-labeled SIINFEKL/H-2K(b) tetramer, CLGGLLTMV/HLA-A*0201 biotinylated monomer, Alexa Fluor 647-labeled CLGGLLTMV/HLA-A*02:01 tetramer, GLCTLVAML/HLA-A*02:01 biotinylated monomer, and Alexa Fluor 647-labeled GLCTLVAML/HLA-A*0201 tetramer, N. Shastri for the B3Z cell line, and G. Freeman for the T2 cell line. We thank Unum Therapeutics for the luciferized Raji cell line and the 19BBz CAR-T cells. We also thank C. Stamoulis from Boston Children′s Hospital and the Harvard Catalyst for her help with statistical analysis; Harvard Catalyst is supported, in part, by the NIH (UL1 TR001102). Lastly, we thank R. Bates, M. Pezone, B. Schultes, L. Edwards, G. Motz, T. Barnitz, T. Hickman, K. McGinness, J. Ritz, M. Maus, T. Snyder, and W.A. Li for valuable scientific discussions.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Representative brightfield microscopy of MSRs. Scale bar, 100 μm. (b) Size distribution of POPC liposomes as measured by DLS. (c) Representative images of lipid-coated MSRs showing the aggregation at lipid-to-MSR 1:20 (w/w). MSRs (left), fluorophore-tagged phospholipid (middle), and co-localization of MSRs and lipid (right). Scale bar, 50 μm. (d) Quantification of lipid retention onto MSRs for different phosphatidylcholines of increasing saturation. C:D are shown in the labels. Retention of different lipid coatings on MSRs over time in PBS (e) and RPMI-1640 containing 10% serum (cRPMI; f). (g) Representative overlaid fluorescence microscopy images of different lipid-coated MSRs, maintained in cRPMI under standard cell culture conditions. Scale bar, 100 μm. (h) Quantification of recombinant human IL-21 (left) and TGF-β (right) released from MSR-SLBs in vitro over time (data points) with sigmoidal curve fitted to data points (IL-21, R2 = 0.99; TGF-β, R2 = 0.97). Approximately 76 ± 4% and 75 ± 5% of the input IL-21 and TGF-β was loaded into MSR-SLBs, respectively. Physical properties of the proteins are listed. Data in (b) represents mean size distribution of three independent samples. Data in (d-f, h) represent mean ± s.d. of three experimental replicates and are representative of at least two independent experiments.
Representative microscopy images of MSR-SLBs not presenting any surface cues (-cue), or surface-presenting αCD3 and αCD28 (APC-ms; +cue), cultured with primary mouse T cells for one day. Images taken at low (left) or high (right) magnification. Cells and material are visible in brightfield images (top) and MSR-SLB lipid coatings are visible in the green channel. Merged images are shown on the bottom. Low magnification scale bar, 500 μm, high magnification scale bar, 100 μm.
(a) Viability of primary mouse T cells isolated from C57BL/6J mice cultured with αCD3/αCD28 Dynabeads of varying doses (D1, D2, D3) or APC-ms of varying formulations (A1, A2, A3, A4; see Table 1), at day 7. (b) Expansion of T cells that were either untreated (mock), or cultured with free cues (50 nM αCD3, 50 nM αCD28, 30 U/ml IL-2), commercial T cell expansion beads (Dynabead, dose D1), IL-2-loaded MSR-SLBs without T cell cues presented on the bilayer surface (MSR-SLB (-cue)), or APC-ms (formulation A1). Curves for mock and free were indistinguishable from the MSR-SLB (-cue) curve. (c) Frequencies of CD4 and CD8 single positive cells (expressed as a ratio of CD4+ to CD8+ cells) isolated from C57BL/6J mice in cultures with Dynabeads or APC-ms, evaluated using FACS. (d) Quantification of CD4+ and CD8+ cells isolated from BALB/c mice after 7 days of culture with APC-ms (A4) or Dynabeads (D2). (e) Representative plots showing CD4 and CD8 expression among T cells derived from C57BL/6J or BALB/c mice cultured with APC-ms (A4) for 7 days. (f) Quantification of Granzyme B positive cells among CD8+ cells expanded with Dynabeads (D1) or APC-ms (A1). (g) Quantification of FoxP3 positive cells among CD4+ cells expanded with Dynabeads (D1) or APC-ms (A1). FACS data gated on Fluorescence Minus One (FMO) controls for each sample, at each timepoint. Data in (b, f-g) represent mean ± s.d. of three experimental replicates. Data in (a, c-d) represent mean ± s.d. of n=4 mice. Data in (a-c, f-g) were conducted using primary T cells from C57BL/6 mice; data in (d) were conducted using primary T cells from BALB/c mice. All data are representative of at least two independent experiments.
(a) Quantification of total amount of αCD3/αCD28 presented on commercial Dynabeads via BCA assay. Approximately 0.46-0.54 ng of αCD3/αCD28 is presented per Dynabead. (b) Amount of αCD3/αCD28 presented on varying Dynabead doses and APC-ms formulations (see Table 1). Data in (a) represents mean ± s.d. of three experimental replicates and are representative of at least two independent experiments.
Supplementary Figure 5 Extended phenotypic characterization of polyclonally expanded primary human T cells
FACS quantification of (a) cell viability and (b) CD62L and CCR7 co-expression among CD3+ cells over time, in samples cultured with either Dynabeads (D1) or APC-ms of varying formulations (see Table 1). Data represents mean ± s.e.m of at least three different donor samples and is representative of at least two independent experiments.
Supplementary Figure 6 Extended characterization of primary human T cells cultured with antigen-specific APC-ms or autologous monocyte-derived dendritic cells
Antigen-specific enrichment (a) and expansion (b) of CLG- or GLC-specific CD8+ T cells cultured with either APC-ms presenting either CLG or GLC or autologous moDCs pulsed with CLG or GLC (right), quantified via FACS. Each line color denotes an individual donor (donors 1, 6, 8, 9, 10, 12), allowing visualization of intra-donor trends. APC-ms consistently promoted antigen-specific T cell expansion but significant donor-to-donor variability in how T cells responded to stimulation was observed. (c) Total expansion of all cells in cultures with APC-ms (left), or autologous moDCs (right). Data are from different donor samples and are representative of at least two independent experiments. APC-ms/CLG, n=4; APC-ms/GLC, n=6; moDC/CLG, n=2; moDC/GLC, n=4.
Supplementary Figure 7 Extended functional characterization of primary human T cells expanded with CLG- or GLC-presenting APC-ms
(a) Quantification of IFNγ secretion by CD8+ T cells cultured with APC-ms presenting either CLG or GLC, following co-culture with T2 cells that were unpulsed (-Peptide), pulsed with CLG (+CLG), or pulsed with GLC (+GLC). (b) Representative plots showing IFNγ and TNFα expression by CD8+ T cells cultured with APC-ms presenting CLG or GLC, following co-culture with T2 cells that were unpulsed (-Peptide; top), pulsed with CLG (+CLG; middle), or pulsed with GLC (+GLC; bottom). Data in (a) represent mean ± s.d. of three experimental replicates and are representative of at least two independent experiments with n=2 donor samples.
Antigen-specific enrichment (a) and expansion (b) of CLG- or GLC-specific CD8+ T cells from CD14+ cell-depleted PBMCs cultured with APC-ms presenting CLG or GLC, quantified via FACS. Data are from different donor samples and are representative of at least three independent experiments. APC-ms/CLG, n=2; APC-ms/GLC, n=4. Only day 14 data is available for the enrichment and expansion of GLC-specific CD8+ T cells.
Supplementary Figure 9 Extended characterization of APC-ms-restimulated 19BBz CAR T cells and their efficacy in a disseminated lymphoma xenograft model
(a) Representative FACS plots showing CD4 and CD8 expression on CD3+ 19BBz T cells at the start of culture (d0), and after 14 days of culture with either Dynabeads (dose D1) or APC-ms (formulation A4). (b) Degradation of APC-ms cultured with mouse T cells, measured via ICP-AES. Si content of APC-ms was below the limit of detection of ICP-AES by day 7. (c-d) Complete IVIS image sets for luciferized Raji-inoculated NSG mice that were mock-treated (RPMI-1640; labelled “M”), or treated with 19BBz T cells expanded for 7 days (c) or 14 days (d) with Dynabeads (labelled “D”) or APC-ms (labelled “A”). The dotted line denotes the average luminescence of mice that were not administered luciferin at the first measured timepoint (d7) and represents baseline. Bioluminescent images (top) and quantification of images (bottom). One animal in these images was excluded from analysis (c, left; denoted by “X”) because it was administered a different dose of 19BBz T cells. For 7 day-expanded 19BBz T cells: mock, n=6; Dynabeads, n=6; APC-ms, n=6. For 14 day-expanded 19BBz T cells: mock, n=6; Dynabeads, n=7; APC-ms, n=7.
Supplementary Figures 1–9 (PDF 1815 kb)
Enrichment of EBV-specific CD8+ 1 T cells following a single stimulation with APC-ms presenting either CLG or GLC. (PDF 124 kb)
Individual rods randomlysettled and stacked to form a 3D scaffold in culture. (WMV 12616 kb)
The large interparticle spaces formed by 3D scaffold were infiltrated by mouse T cells. (WMV 10742 kb)
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Cheung, A., Zhang, D., Koshy, S. et al. Scaffolds that mimic antigen-presenting cells enable ex vivo expansion of primary T cells. Nat Biotechnol 36, 160–169 (2018). https://doi.org/10.1038/nbt.4047
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