Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8+ T Cell Responses with Antitumor Activity

Abstract

CD8⁺ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8⁺ T cell responses, therapeutic approaches to generate Ag-specific CD8⁺ T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8⁺ T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ∼1000-fold more potent at eliciting CD8⁺ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8⁺ T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8⁺ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8⁺ T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8⁺ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.

FIGURE 1.

Microfluidic squeezing enhances Ag presentation by professional APCs. (A) Cells are squeezed in the presence of Ag in a microfluidic chip that creates temporary pores in the cells, allowing Ag to diffuse into the cytosol, from which it can be presented on MHC-I. (B–E) Mouse BMDCs were incubated with varying concentrations of fluorescently labeled OVA for 30 min at 37°C (Incub. Ctrl) or squeezed with the same concentrations (Squeeze). (B) OVA MFI for Incub. Ctrl and Squeeze groups at varying OVA concentrations. (C) Incub. Ctrl and Squeeze BMDCs were cocultured with OT-I CD8⁺ T cells for 24 h, and CD69 staining on the OT-I cells was assessed by flow cytometry. (D and E) Mouse BMDCs were incubated with varying concentrations of fluorescently labeled OVA for 30 min at 37°C (Incub. ctrl) or were squeezed with the same concentrations (Squeeze). Incub. ctrl and squeezed BMDCs were incubated for 4 h at 37°C and subsequently stained with 25-D1.16 Ab and analyzed by flow cytometry. Representative plots for indicated concentrations are shown in (D). Summary data for all concentrations are shown in (B). Data are representative of two independent experiments.

FIGURE 2.

Microfluidic squeezing enables Ag presentation in vivo by diverse immune cell types and requires adjuvant. (A) Mice were left untreated or immunized with 1 × 10⁶ B cells treated as indicated. Spleens were harvested 7 d later and restimulated with SIINFEKL peptide to assess IFN-γ–producing CD8⁺ T cells by flow cytometry. N = 5 mice per group. (B) Mice were left untreated or were immunized with 1 × 10⁶ T cells treated as indicated. Spleens were harvested 7 d later and restimulated with SIINFEKL peptide to assess IFN-γ–producing CD8⁺ T cells by flow cytometry. N = 5 mice per group. (C) Mice were left untreated or were immunized with 1 × 10⁶ murine PBMC surrogate cells treated as indicated. All groups were matured with CpG before injection. Spleens were harvested 7 d later and restimulated with SIINFEKL peptide to assess IFN-γ–producing CD8⁺ T cells by flow cytometry. n = 5 mice per group. (D) Mouse splenocytes were left untreated (NC), incubated at room temperature with fluorescent 3 kDa dextran (Incub. ctrl), or squeezed in the presence of fluorescently labeled 3 kDa dextran (Squeeze). Viability (left) and dextran delivery (right) of each cell type were immediately determined by flow cytometry. (E) Mouse splenocytes were squeezed without cargo (Squeeze only) or squeezed in the presence of OVA (Squeeze + OVA). At the indicated time points, cells were stained with the 25-D1.16 Ab to assess SIINFEKL presentation on H-2Kb and phenotypic markers to define cell subsets. ***p < 0.001.

FIGURE 3.

Ag presentation by squeezed immune cells is direct and primes antitumor immunity. (A) A total of 2.5 × 10⁶ CFSE-labeled CD8⁺ OT-I cells were transferred into either WT or MHC-I^−/− (knockout [KO]) mice. A few hours later, 5 × 10⁶ splenocytes squeezed in the presence of OVA and incubated for 4 h with CpG were injected i.v. After 3 d, lymph nodes were harvested, and proliferation of OT-I cells was assessed by CFSE dilution. n = 5 mice per group. (B) Mouse B cells were not in contact with OVA (NC), incubated at room temperature with OVA (Incub. ctrl), squeezed in the presence of OVA (Squeeze), or pulsed with SIINFEKL peptide for 1 h at 37°C. A total of 5 × 10⁶ B cells were coinjected with 3 µg LPS to immunize mice that had also received 2.5 × 10⁶ CFSE-labeled OT-I CD8⁺ T cells. CFSE dilution by the OT-I cells was assessed 3 d after immunization in lymph nodes. n = 5 mice per group. (C) Mouse B cells were not in contact with gp100 SLP (NC), incubated at room temperature with gp100 SLP (Incub. ctrl), squeezed in the presence of the gp100 SLP (Squeeze), or pulsed with short peptide for 1 h at 37°C (PP). A total of 5 × 10⁶ B cells were coinjected with 3 µg LPS to immunize mice that had also received 2.5 × 10⁶ CFSE-labeled pmel CD8⁺ T cells. CFSE dilution by the pmel CD8⁺ T cells was assessed 3 d after immunization. n = 5 mice per group. (D) Mouse splenocytes were squeezed with or without OVA. After squeezing, indicated populations of cells were magnetically separated and cocultured with OT-I TCR-transgenic cells overnight. CD69 MFI on OT-I cells was assessed by flow cytometry. Data are representative of two independent experiments. (E) Mice were left untreated or immunized on day −14 and day −7 with 1 × 10⁶ murine PBMC surrogate cells incubated or squeezed with OVA protein and matured with CpG. On day 0, mice were s.c. implanted with E.G7-OVA and monitored for tumor growth. ***p < 0.001.

FIGURE 4.

Microfluidic squeezing enables MHC-I presentation of Ags by diverse human immune cells. (A) Immune cells from healthy human donors were squeezed with an SLP containing the HLA-A2–restricted CMV pp65 epitope, NLVPMVATV, and incubated overnight with HLA-A2–restricted pp65 responder CD8⁺ T cells. Supernatants were collected for IFN-γ analysis by ELISA. (B–E) Human moDCs (n = 3), B cells (n = 4), T cells (n = 3), or PBMCs (n = 3) were left untreated, incubated with pp65 SLP, squeezed without Ag, squeezed with pp65 SLP, or pulsed with the minimal epitope NLVPMVATV (Pep. Pul.). IFN-γ production from HLA-A2–restricted pp65 responder CD8⁺ T cells after overnight incubation is shown. (F) Human B cells were squeezed or incubated with a pp65 SLP containing the HLA-A2–restricted epitope, a pp65 SLP containing an HLA-B35–restricted epitope, or squeezed with both. IFN-γ production from HLA-A2– or HLA-B35–restricted pp65 responder CD8⁺ T cells is shown. (G–I) For research scale, cells were isolated from human blood, and 2 × 10⁶ cells were squeezed. For manufacturing scale, cells were isolated from apheresis products, and the isolated product was squeezed. Viability and delivery were determined by flow cytometry immediately after delivery. Each dot is representative of a single donor. Delivery was determined on the basis of cells that were not incubated with dextran. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5.

Squeezed cells prime antitumor activity in a mouse model of HPV16⁺ tumors. (A) Mice were left untreated (Untr.) or immunized with T cells squeezed with the E7 SLP containing the H2-D^b-restricted epitope RAHYNIVTF and coinjected with CpG. Seven days later, E7-specific responses were measured in the spleen by intracellular cytokine staining (ICS). (B) Mice were prophylactically immunized with 1 × 10⁶ T cells squeezed with E7 SLP and coinjected with CpG on day −7. On day 0, mice were challenged s.c. with TC-1. n = 10 mice per group. (C) Ten days after TC-1 inoculation, mice were immunized with 1 × 10⁶ T cells squeezed with E7 SLP with or without coinjection of CpG. Mean tumor volume + SEM is shown. n = 10 mice per group. (D) Mice were left untreated or immunized with 5 × 10⁶ B cells squeezed with E7 SLP and matured with CpG. Seven days later, E7-specific responses were measured by ICS. (E) Mice were immunized with 5 × 10⁶ B cells squeezed with E7 SLP and matured with CpG overnight or left untreated. Seven days later, mice were challenged s.c. with TC-1. Survival is shown. n = 10 mice per group. (F) Mice were immunized 14 d after TC-1 implantation using 5 × 10⁶ B cells pulsed with minimal epitope (Min. Epi.), RAHYNIVTF, or squeezed with E7 SLP and matured with CpG overnight. An additional group of mice was immunized and boosted on days 14 and 28 s.c. with 150 µg E7 SLP and 50 µg CpG. Mean tumor volume + SEM is shown. n = 10 mice per group. (G) Mice were immunized as indicated 14 d after TC-1 implantation. Seven days later, tumors were harvested for analysis by flow cytometry. Mean ± SD is shown. (H) Mice were left untreated or immunized with 1 × 10⁶ splenocytes squeezed with E7 SLP and matured overnight with CpG. Seven days later, E7-specific responses were measured in the spleen by ICS. (I) Mice were immunized on days −14 and −7 with 1 × 10⁶ M-SQZ-PBMC-HPV or left untreated. On day 0, TC-1 tumors were implanted, and tumor growth was monitored. Sixty days after inoculation, immunized mice were rechallenged with TC-1 on the opposite flank, and a new cohort of untreated mice was introduced. (J) Ten days after TC-1 implantation, mice were immunized with 1 × 10⁶ splenocytes squeezed with E7 SLP that had been matured with CpG for 4 h or incubated without CpG. Tumor volume + SEM is shown. n = 10 mice per group. (K) Mice were primed with M-SQZ-PBMC-HPV at a dose of 1 × 10⁶ or 1 × 10⁵ cells on day 10. An additional group of mice was primed on day 10 and boosted two more times on days 17 and 24 (P/B/B). (L) Mice were immunized with 1 × 10⁶ M-SQZ-PBMC-HPV or s.c. 150 μg E7 SLP with 50 μg CpG 16 d after TC-1 implantation. Twelve days later, tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Mean percentage ± SD of indicated populations is shown. *p < 0.05, **p < 0.01, ***p < 0.001.