Surface-Functionalized Microgels as Artificial Antigen-Presenting Cells to Regulate Expansion of T Cells

Abstract

Artificial antigen-presenting cells (aAPCs) are currently used to manufacture T cells for adoptive therapy in cancer treatment, but a readily tunable and modular system can enable both rapid T cell expansion and control over T cell phenotype. Here, it is shown that microgels with tailored surface biochemical properties can serve as aAPCs to mediate T cell activation and expansion. Surface functionalization of microgels is achieved via layer-by-layer coating using oppositely charged polymers, forming a thin but dense polymer layer on the surface. This facile and versatile approach is compatible with a variety of coating polymers and allows efficient and flexible surface-specific conjugation of defined peptides or proteins. The authors demonstrate that tethering appropriate stimulatory ligands on the microgel surface efficiently activates T cells for polyclonal and antigen-specific expansion. The expansion, phenotype, and functional outcome of primary mouse and human T cells can be regulated by modulating the concentration, ratio, and distribution of stimulatory ligands presented on microgel surfaces as well as the stiffness and viscoelasticity of the microgels.

Keywords: T cell activation; antigen‐specific T cell expansion; granular hydrogels; microgels; surface functionalization; viscoelasticity.

Figure 1.

Fabrication and characterization of microgels and granular hydrogels. a) Schematic representation of microgel preparation using microfluidic emulsion. Alginate microgels were crosslinked by norbornene-tetrazine click chemistry. b) Phase-contrast image of alginate microgels crosslinked by norbornene-tetrazine click chemistry. Scale bar: 100 μm. c) Elastic moduli of 2 wt% alginate microgels containing different Nb/Tz ratios as measured by AFM. All the data sets are significantly different (**** P < 0.0001) except the two compared in the figure. d) Schematic representation of microgel assembly and jamming. Concentrated microgels were loaded on a membrane filter to remove continuous phase by centrifugation, which resulted in jamming of microgels. e) Void space in granular hydrogel calculated from 2D confocal slices as a function of increasing centrifugation time. In [c] and [e], values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001 and NS, not significant.

Figure 2.

Surface functionalization of microgels. a) Schematic representation of PDL and subsequent alginate coating on the surface of microgels. b) Confocal images of both PDL and alginate coatings. Scale bar: 100 μm. c) Confocal image of alginate-Rhodamine B coating on microgel surface at 100x magnification; thickness = 0.74 ± 0.11 μm. Scale bar: 20 μm. d) Change in quantity of alginate coating on microgel surface over 3 weeks in beads buffer. e) Change in quantity of alginate coating on microgel surface over 1 week in T cell culture media. f) Density of alginate polymer coated on microgel surface as a function of alginate concentration in coating solutions. g) Confocal image of azide-coated microgels labelled with Rhodamine-DBCO. Scale bar: 200 μm. h) Confocal images of alginate-Rhodamine B coated on the surface of microgels formed from hyaluronic acid, gelatin and an alginate/collagen interpenetrating network. Scale bar: 100 μm. i) Confocal images of alginate microgel presenting tetrazine functional groups coated with alginate-sulfoCy5. Microgel core in red, free tetrazine in green and polymer coating in blue. Scale bar: 20 μm. j) Quantification of fluorescent intensity of Rhodamine B (red), FITC (green) and sulfoCy5 (blue) as a function of distance from microgel surface. In [d]-[f], values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001.

Figure 3.

Polyclonal and antigen-specific activation of primary mouse T cells. a) Schematic representation of modification of αCD3 and αCD28 antibodies on microgel surface by coating microgels with azide-modified alginate and conjugating antibodies using azide-DBCO click chemistry. b) UV-vis absorption spectra of unmodified αCD3, DBCO-modified αCD3 and DBCO model compounds. c) Carboxifluorescein diacetate succinimidyl ester (CFSE) histogram evaluated using FACS flow cytometry indicating the proliferation profile of stimulated CD4+ T cells. d) Percentage of proliferating CD4+ T cells when cultured with Dynabeads or microgels of different formulations. e) Representative phase contrast images of primary mouse CD4+ T cells cultured with blank microgels, microgels conjugated with anti CD3/CD28 over the entire microgel and microgels functionalized with anti CD3/CD28 on the surface as shown by representative images using phase contrast. Scale bar: 100 μm. f) Schematic representation of modification of MHC-Ⅰ and αCD28 on microgel surface. Microgels were first coated with biotin-modified alginate, reacted with streptavidin and then conjugated with ligands using biotin-streptavidin interaction. g) CFSE histogram evaluated using FACS flow cytometry indicating the proliferation profile of stimulated antigen-specific CD8+ T cells. h) Representative plots and i) quantification showing enrichment of live CD8+ cells specific for SIINFEKL peptides when mixed CD8+ T cells were cultured with Dynabeads or MHC-Ⅰ/antigen functionalized microgels. j) Fold expansion of CD8+ T cells specific for SIIFEKL peptide cultured with Dynabeads or MHC-Ⅰ functionalized microgels. In [d], [i] and [j], values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001 and NS, not significant.

Figure 4.

Polyclonal mouse T cell expansion (CD4+ and CD8+ co-culture) by varying biochemical properties of microgels. a) Expansion of primary mouse T cells (inlcuding CD4+ and CD8+ T cells), b) CD4/CD8 ratio of CD4+ and CD8+ single-positive cells, c) CD44 and CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of overall surface antibody density or Dynabeads on Day 3. αCD3/ αCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0. d) Quantification of in vitro killing of OVA-expressing B16-F10 target cells by CD8+ OT-I T cells that were co-cultured with microgels as a function of overall surface antibody density. Effector/target cell ratios = 10:1. e) Expansion of primary mouse T cells (inlcuding CD4+ and CD8+ T cells), f) CD4/CD8 ratio of CD4+ and CD8+ single-positive cells, g) CD44 and CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of αCD3/ αCD28 ratio on Day 3. Overall antibody density = 0.4 μg/cm², CD4/CD8 ratio = 1 on Day 0. h) Schematic representation of a single type microgel coated with antibodies (medium purple, left) and a mixture of microgels coated with antibodies (dark purple, right) and without antibodies (light purple, right). i) Expansion of primary mouse T cells, j) CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were co-cultured with a single type microgel or mixed microgels at the same overall surface antibody density on Day 3. αCD3/ αCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0. k) Schematic representation of modification of αCD3 and αCD28 antibodies and IL-2 on microgel surface. l) Expansion of primary mouse T cells, m) CD4/CD8 ratio of CD4+ and CD8+ single-positive cells that were co-cultured with microgels as a function of IL-2 density on Day 3. Overall antibody density = 0.8 μg/cm², αCD3/ αCD28 ratio = 1, CD4/CD8 ratio = 1 on Day 0. In [d]-[f], [i], [j], [l] and [m], values represent mean ± s.d. an ordinary one-way ANOVA with post hoc Tukey’s multiple comparisons was used. *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001 and NS, not significant.

Figure 5.

Polyclonal mouse T cell expansion (CD4+ and CD8+ co-culture) while varying the physical properties of microgels. a) Expansion of primary mouse T cells and b) CD44/CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of stiffness for 3 days. c) Expansion of primary mouse T cells and d) CD44/CD62L expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with elastic or viscoelastic microgels for 3 days. Overall antibody density = 0.4 μg/cm², αCD3/ αCD28 ratio = 1 in all studies.

Figure 6.

Polyclonal human T cell expansion (mixture of CD4+ and CD8+) by varying biochemical properties of microgels. a) Expansion of primary human T cells that were co-cultured with microgels as a function of overall surface antibody density on Day 6. b) CD25 expression by live CD4+ (left) or CD8+ (right) T cells that were co-cultured with microgels as a function of overall surface antibody density on Day 6. c) CD4/CD8 ratio of cells cultured with microgels as a function of overall surface antibody density on Day 6. d) CD45RA and CCR7 expression, e) CD39 expression by live CD4+ (left) or CD8+ (right) T cells that were cultured with microgels as a function of overall surface antibody density on Day 6.