Ex vivo expansion and hydrogel-mediated in vivo delivery of tissue-resident memory T cells for immunotherapy

Abstract

Tissue-resident memory T (T_RM) cells preferentially reside in peripheral tissues, serving as key players in tumor immunity and immunotherapy. The lack of effective approaches for expanding T_RM cells and delivering these cells in vivo hinders the exploration of T_RM cell-mediated cancer immunotherapy. Here, we report a nanoparticle artificial antigen-presenting cell (nano-aAPC) ex vivo expansion approach and an in vivo delivery system for T_RM cells. Using the nano-aAPC platform, we expanded functional antigen-specific murine and human T_RM-like CD8⁺ T cells ex vivo. We also developed an injectable macroporous hyaluronic acid (HA) hydrogel to deliver T_RM-like cells. T_RM-like cells delivered in the optimized HA hydrogel trigger robust local and systemic antitumor immunity and show synergistic effects with anti-PD-1 treatment. Our findings suggest that nano-aAPC-induced T_RM-like cells, coupled with a hydrogel delivery system, offer an efficient way to advance the understanding of T_RM cell-mediated cancer therapy.

Fig. 1.. Schematics of T_RM-like cell generation stimulated and expanded by nano-aAPCs conjugated with pMHC-I and anti-CD28 antibodies in the presence of IL-2, IL-15, and TGF-β, and delivery of T_RM-like cells by HA hydrogel in vivo.

Created with BioRender.com.

Fig. 2.. Nano-aAPCs with IL-2, IL-15, and TGF-β induce functional antigen-specific mouse CD8⁺ T cells with T_RM phenotype and transcriptional profile.

(A) Fold expansion of PMEL and OT-I CD8⁺ T cells expanded by nano-aAPCs in the presence of various cytokine mixes over 6 days. Each bar represents means ± SEM. n = 3. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. ***P < 0.001. (B) Expression of T_RM marker (CD69 and CD103) on PMEL and OT-I CD8⁺ T cells on day 6. (C) Expression of homing marker (CD62L) on PMEL and OT-I CD8⁺ T cells on day 6. (D) Principal components analysis (PCA) of top 1000 differentially expressed gene (DEG) expression data from OT-I CD8⁺ T cells expanded by aAPCs in the presence of IL-2 (T_Eff) or IL-2, IL-15, and TGF-β (T_RM-like). (E) Heatmap of T_RM-related signature genes expression in OT-I CD8⁺ T cells expanded by aAPCs in the presence of IL-2 (T_Eff) or IL-2/IL-15/TGF-β (T_RM-like) (F) Pie charts showing the polyfunctionality profile of PMEL and OT-I CD8⁺ T cells expanded in the medium containing nano-aAPCs, IL-2, IL-15, and TGF-β. Pie charts represent the fractions of expanded T cells secreting none or any (1, 2, or 3) of the three cytokines IFN-γ, TNF-α, and IL-2.

Fig. 3.. Injectable HA macroporous hydrogel–released T_RM-like cells display robust antigen-specific cytolytic activity.

(A) Scanning electron microscopy (SEM) image of HA hydrogel showing macroporous structure throughout the hydrogel; scale bars, 250 μm. (B) Pore sizes of HA hydrogel. n = 49. Two tailed unpaired Student’s t test with Welch’s correction. ***P < 0.001. (C) The confocal microscopy three-dimensional (3D) reconstruction images of activated PMEL CD8⁺ T cells migration in different HA hydrogels; scale bars, 500 μm. (D) Quantitative analysis of infiltrating distance of CD8⁺ T cells in the HA hydrogel. (E) Schematic of the experimental setup to measure T cell release from HA hydrogel. Created with BioRender.com. (F) Percentage of cells released from HA hydrogel. (G) Schematic of the experimental setup to access antigen-specific killing of adoptively transferred CD8⁺ T cells. sc, subcutaneous; iv, intravenous. PMEL T_RM-like cells induced in vivo killing in the (H) spleens and (I) draining lymph nodes. Each bar represents means ± SEM. n = 5. One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 and **P < 0.01.

Fig. 4.. T_RM-EH exhibits higher antitumor efficacy than intravenously administered T_RM-like cells.

(A) Schematic of the experimental design. Mice were administered either naïve OT-I CD8⁺ T cells (intravenous), OT-I T_RM CD8⁺ T cells (intravenous), or OT-I T_RM-EH (subcutaneous) 1 day before subcutaneous MC38-OVA tumor cell injection. (B) MC38-OVA tumor growth curves with means ± SEM. n = 5 to 6. Two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 and ***P < 0.001. (C) Mouse survival curve. n = 5 to 6. Log-rank test. **P < 0.01. (D) Schematic of the experimental design. Mice were administered either naïve OT-I CD8⁺ T cells (intravenous), OT-I T_RM-like CD8⁺ T cells (intravenous), or OT-I T_RM-EH (subcutaneous) 1 day before subcutaneous MC38-OVA tumor cell injection. On day 13, tumors were isolated, and tumor-infiltrating immune cells were analyzed. Quantification of total (E) CD45⁺, (F) CD8⁺, (G) CD8⁺ CD45.2⁺, (H) CD4⁺, (I) natural killer (NK), (J) macrophages, and (K) CD11b^– dendritic cells (DC) in the tumor is shown. Each bar represents means ± SEM. n = 3 to 4. One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 and **P < 0.01. n.s., not significant.

Fig. 5.. T_RM-EHs elicited robust local and systemic immune responses in a mouse colon carcinoma model.

(A) Schematic of the experimental design. OT-I T_RM-EHs were subcutaneously injected on the same or opposite side of the MC38-OVA tumor 5 days after tumor inoculation. (B) MC38-OVA tumor growth curves with means ± SEM. n = 5 to 6. Two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Mice survival curve. n = 5. Log-rank test. *P < 0.05 and **P < 0.01. (D) Schematic of the experimental design. OT-I T_RM-EHs were subcutaneously injected on the same or opposite side of the MC38-OVA tumor 5 days after tumor inoculation. On day 12, tumors were isolated, and tumor-infiltrating immune cells were analyzed. (E) Quantification of total CD45⁺, CD8⁺, CD45.2⁺, CD4⁺, and NK cells in the tumor is shown. n = 9. One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 and ***P < 0.001. (F) Schematic of the experimental design. OT-I CD8⁺ T_RM-EHs were subcutaneously injected on the same side of the MC38-OVA tumor 5 days after tumor cell injection. On day 14, we isolated the tumors and analyzed tumor-infiltrating immune cells. Quantification of total (G) CD45⁺, CD8⁺ (H) CD4⁺, NK cells, CD11b⁺ dendritic cells, and macrophages in the tumors is shown. Each bar represents means ± SEM. n = 8. Two-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.. T_RM-EHs synergized with PD-1 blockade therapy in a mouse melanoma model.

(A) Schematic of the experimental design. Mice were subcutaneously injected with B16-OVA tumor cells. OT-I T_RM-EHs were subcutaneously injected 5 days after tumor cell injection. The T_RM-EHs were injected on the same flank as the tumor, followed by three anti–PD-1 injections on days 5, 8, and 12. (B) B16-OVA tumor growth curve with means ± SEM. n = 5. Two-way ANOVA with Tukey’s multiple comparisons test. ***P < 0.001. (C) Mouse survival curve. n = 5. Log-rank test. *P < 0.05 and **P < 0.01. ip, intraperitoneal.

Fig. 7.. Nano-aAPCs induce functional antigen-specific human CD8⁺ T cells with T_RM-like phenotype.

(A) Cell number of MART-1⁺ CD8⁺ T cells expanded by nano-aAPCs in the presence of various cytokine mixes on days 0, 7, and 14. Each bar represents means ± SEM. n = 3 donors. (B) Fold expansion of MART-1⁺ CD8⁺ T cells expanded by nano-aAPCs in the presence of various cytokine mixes over 14 days. Each bar represents means ± SEM. n = 3 donors. (C) Tetramer staining of MART-1⁺ CD8⁺ T cells expanded by nano-aAPCs in the presence of various cytokine mixes on day 14 from three healthy donors. (D) Granzyme B expression of MART-1⁺ CD8⁺ T cells on day 14. Mean fluorescence intensity of (E) CD69 expression, (F) CD103 expression, and (G) CD62L expression of MART-1⁺ CD8⁺ T cells on day 14. Each bar represents means ± SEM. n = 3 donors. One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 and ***P < 0.001.