Efficient ex vivo induction of T cells with potent anti-tumor activity by protein antigen encapsulated in nanoparticles

Abstract

Protein antigen (Ag)-based immunotherapies have the advantage to induce T cells with a potentially broad repertoire of specificities. However, soluble protein Ag is generally poorly cross-presented in MHC class I molecules and not efficient in inducing robust cytotoxic CD8(+) T cell responses. In the present study, we have applied poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) which strongly improve protein Ag presentation by dendritic cells (DC) in the absence of additional Toll-like receptor ligands or targeting devices. Protein Ag-loaded DC were used as antigen presenting cells to stimulate T cells in vitro and subsequently analyzed in vivo for their anti-tumor effect via adoptive transfer, a treatment strategy widely studied in clinical trials as a therapy against various malignancies. In a direct comparison with soluble protein Ag, we show that DC presentation of protein encapsulated in plain PLGA-NP results in efficient activation of CD4(+) and CD8(+) T cells as reflected by high numbers of activated CD69(+) and CD25(+), interferon (IFN)-γ and interleukin (IL)-2-producing T cells. Adoptive transfer of PLGA-NP-activated CD8(+) T cells in tumor-bearing mice displayed good in vivo expansion capacity, potent Ag-specific cytotoxicity and IFN-γ cytokine production, resulting in curing mice with established tumors. We conclude that delivery of protein Ag through encapsulation in plain PLGA-NP is a very efficient and simple procedure to stimulate potent anti-tumor T cells.

Fig. 1

Efficient MHC class I and class II presentation of OVA Ag incorporated in PLGA-NP. a D1 cells were pulsed for 24 h with titrated amounts (μM) of OVA, either in soluble form sOVA or encapsulated in PLGA-NP (PLGA-OVA). MHC class I presentation was detected by co-culture with H-2K^b/SIINFEKL-specific B3Z CD8⁺ T cells; b D1 cells were pulsed for 24 h with 0.25 μM OVA in PLGA-OVA, empty PLGA-NP or a mixture of empty PLGA-NP with 0.25 μM sOVA washed and co-cultured with B3Z CD8⁺ T cells to assess MHC class I Ag presentation; c D1 cells were pulsed for 24 h with titrated amounts of PLGA-OVA or sOVA, washed and co-cultured with I-A^b/ISQAVHAAHAEINEAGR-specific OT-IIZ CD4⁺ T cells to assess MHC class II Ag presentation. BMDC were loaded with titrated amounts of PLGA-OVA or sOVA. Ag-loaded DC were subsequently used to activate naïve OT-I (d) or OT-II T (e) cells for 72 h. T cell proliferation was measured in triplicate by 3[H]-thymidine uptake. Data shown are means of triplicate measurements ± SD from one representative example out of at least three independent experiments

Fig. 2

DC pulsed with PLGA-OVA, but not sOVA, induce strong activation of T cells. D1 cells were pulsed for 24 h with titrated amounts of sOVA or PLGA-OVA. Ag-loaded DC were washed to remove excess Ag, and co-cultured for an additional 24 h with OT-I or OT-II splenocytes. Cells were harvested and analyzed by flow cytometry for the cell surface expression of CD69 on CD8⁺ T (a) cells and CD4⁺ T cells (b); c Expression of CD25 and CD69 was analyzed on CD8⁺ T cells which were stimulated for 24 h with DC which were loaded with either PLGA-OVA or sOVA. Immature DC without Ag served as negative control. Intracellular production of IL-2 and IFN-γ by CD8⁺ T cells (d); and CD4⁺ T (e) cells was analyzed by flow cytometry after 24 h stimulation with DC pulsed with titrated amounts of PLGA-OVA or sOVA. One representative experiment out of three independent experiments is shown. Data shown are means of triplicate measurements ± SD

Fig. 3

Enhanced in vivo cytotoxicity of ex vivo PLGA-OVA-stimulated CD8⁺ T cells. a Mice were transferred with purified CD8⁺ OT-I T cells which were in vitro stimulated with D1 cells loaded with PLGA-OVA and sOVA. Differentially, CFSE-labeled SIINFEKL-peptide loaded and control-target cells were i.v. administered. After 18 h, the spleens from recipient animals were harvested and analyzed by flow cytometry for percentage of specific killing of target cells; b experiment was performed twice and averages ± SEM of n = 7 mice for each condition are shown in bar graphs

Fig. 4

CD8⁺ T cells primed by PLGA-OVA-loaded DC eradicate established tumors. Animals were inoculated s.c. on the right flank with 2 × 10⁵ B16-OVA tumor cells and rested for 1 week followed by a single i.v. injection of 2.5 × 10⁶ purified CD8⁺ T cells which were ex vivo stimulated with Ag-loaded DC as described above. Tumor growth was monitored in individual animals treated with DC/PLGA-OVA-induced CD8⁺ T cells (a; n = 12 animals), DC/sOVA-induced CD8⁺ T cells (b; n = 12 animals) or in untreated animals (c; n = 8 animals). Insert in (a) represents tumor growth curves in the initial 19 days after tumor challenge; d Animal survival per group was assessed, and differences between the different groups were calculated using Log-rank (Mantel-Cox) test. p < 0.001 for animals treated with DC/PLGA-OVA compared to DC/sOVA-induced CD8⁺ T cells. p < 0.0001 for animals treated with DC/PLGA-OVA compared to untreated animals. NS = p > 0.05 for animals treated with DC/sOVA compared to untreated animals

Fig. 5

PLGA-OVA stimulated CD8⁺ T cells expand and persist in the peripheral blood and have higher capacity to produce IFN-γ. a Tumor-bearing animals received a single i.v. injection of CD8⁺ T cells stimulated with PLGA-OVA-loaded DC. Tail vein blood samples were taken on day 10 after adoptive transfer of the CD8⁺ T cells, and numbers of CD8⁺Thy1.1⁺ T cells were measured by flow cytometry; b In vivo persistence of i.v. transferred CD8⁺Thy1.1⁺ T cells in tumor-bearing animals was monitored for 4 weeks in blood on days 17 and 31; c Intracellular IFN-γ production by CD8⁺ T cells was analyzed on days 10, 17 and 31 after adoptive transfer; d Memory phenotype of transferred DC/PLGA-OVA in vitro stimulated CD8⁺Thy1.1⁺ T cells was determined by analysis of CD44 and CD62L surface expression. Results shown are averages ± SEM from n = 3–12 mice per group, dependent on the number of animals alive at each time-point post-tumor challenge. *p < 0.05 for animals treated with DC/PLGA-OVA compared to DC/sOVA-induced CD8⁺ T cells using a unpaired student’s t test