Immune stimulatory antigen loaded particles combined with depletion of regulatory T-cells induce potent tumor specific immunity in a mouse model of melanoma

Abstract

Anti-tumor vaccines capable of activating both CD4 and CD8 T cells are preferred for long lasting T cell responses. Induction of a tumor-specific T-cell response can be induced by tumor vaccines that target innate immunity. The ensuing T-cell response depends on efficient antigen presentation from phagocytosed cargo in the antigen presenting cell and is augmented by the presence of Toll-like receptor (TLR) ligands within the cargo. Biodegradable polymers are useful for vaccine delivery in that they are phagocytosed by antigen presenting cells (APCs) and could potentially be loaded with both the antigen and immune stimulatory TLR agents. This study was undertaken to evaluate the effect of poly lactic-co-glycolic acid (PLGA) polymer particles loaded with antigenic tumor lysate and immune stimulatory CpG oligonucleotides on induction of tumor specific immunity in a mouse model of melanoma. We found that after delivery, these immune stimulatory antigen loaded particles (ISAPs) efficiently activated APCs and were incorporated into lysosomal compartments of macrophages and dendritic cells. ISAP vaccination resulted in remarkable T cell proliferation, but only modestly suppressed tumor growth of established melanoma. Due to this discordant effect on tumor immunity we evaluated the role of regulatory T cells (Treg) and found that ISAP vaccination or tumor growth alone induced prolific expansion of tumor specific Treg. When the Treg compartment was suppressed with anti-CD25 antibody, ISAP vaccination induced complete antigen-specific immunity in a prophylactic model. ISAP vaccination is a novel tumor vaccine strategy that is designed to co-load the antigen with a TLR agonist enabling efficient Ag presentation. Targeting of T-reg expansion during vaccination may be necessary for inducing effective tumor-specific immunity.

Fig. 1

Microparticle preparation and release profiles: a Microparticles were prepared from poly lactic and glycolic acid (PLGA) using the water-in-oil-in-water double emulsion solvent evaporation method. Scanning electron microscopy analysis showed that particles had a smooth morphology and spherical shape. b Release profiles of CpG and tumor lysaste were measured using UV spectrophotometry at 260/280 nm and the micro-BCA protein assay, respectively. Release profiles of both CpG and tumor lysate showed a burst followed by a more sustained release profile

Fig. 2

Light microscopy of a, empty microparticles and fluorescent microscopy of b microparticles loaded with rhodamine. Confocal fluorescent microscopy of RAW264.7 cells treated with microparticles loaded with ovalbumin bound to flouroscein (green) and subsequently counterstained with fluorescent dyes specifically targeting, c the lysosome (red) and d nucleus (blue) and lysosome (red). Confocal microscopy revealed localization of the microparticles within the lysosomes of the macrophages (color available only in online publication)

Fig. 3

Macrophage stimulation by ISAPs. Primary mouse bone-marrow derived macrophages were treated with various components of the microparticles (MP) and their effects on a surface marker expression and b secretion of IL-12 p40 and TNF-α were compared. These studies demonstrate the potent immuno-stimulatory effect of ISAPs on primary macrophages. The immuno-suppressive cytokine IL-10 was not detected in supernatants from any of the groups (data not shown). MP empty microparticle only, MP-CpG MP containing CpG only, MP-B16 MP containing B16 tumor lysate only and ISAP: MP containing CpG and B16 tumor lysate. Studies were performed in triplicate and repeated three times with similar results

Fig. 4

Uptake of microparticles by splenic DCs. Empty PLGA microparticles (MP) or MP loaded with Rhodamine were administered IP into mice. Mice were sacrificed at 1, 3 and 7 days following their administration and splenocytes were harvested for flow cytometry and counter stained for CD11c. a Control empty microparticles, b 1 day, c 3 days and d 4 days following administration of rhodamine loaded MPs. The experiments show that IP administered MPs are taken up and found in splenic DCs following administration for at least 4 days with the highest concentration on day 1. Percentages noted are % of CD11c + (DC) splenocytes. The right lower quadrant probably represents CD11c-macrophages (and possible but unlikely B-cells) that also took up the rhodamine positive MPs. Although B-cells also phagocytose particles, the particle size of the ISAP is predictably too large for B-cells to ingest

Fig. 5

T-cell proliferation following ISAP vaccination. Splenocytes harvested from naïve mice, mice vaccinated with microparticles only or mice vaccinated with various components of the immune stimulatory antigen loaded particles were re-stimulated with tumor lysate and incubated with CFSE. After 7 days of incubation, cells were harvested and stained with anti-CD8 Ab, and then subjected to flow cytometry analysis. Proliferating cells are defined by the dilutional effect of CFSE staining in the T-cells. ISAP vaccination induced significant proliferation of CD8 T-cells when both CpG and tumor lysate (TL) were loaded. (MP empty microparticle only, MP-CpG MP containing CpG only, MP-Alum-TL MP containing alum and B16 tumor lysate, MP-GM-TL MP containing GM-CSF and B16 tumor lysate, MP-CpG-TL (ISAP) MP containing CpG and B16 tumor lysate and MP-GM-CpG-TL MP containing GM-CSF plus CpG plus tumor lysate

Fig. 6

Tumor specific expansion of Treg cells: mice were inoculated with B16 melanoma (1 × 10⁶) and when tumors were either 9 or 18 mm in a single dimension, mice were sacrificed and spleens were harvested. The compartment of CD4 + CD25 + Foxp3 + Treg cells was determined by flow cytometry following 5 days of splenocyte culture with irradiated tumor cells. The percentage of Treg cells of CD4+ gated cells or total splenocytes (in parentheses) proliferated expansively following culture and increased as the tumor size grew larger. (Fig. 6a) In another experiment splenocytes were harvested from a mouse bearing a large tumor and cultured with various lysate antigens. The Treg expansion observed was tumor specific as it only occurred when the splenocytes were cultured with the B16 tumor antigen (lysate) and not with other unrelated tumor antigens (Neuro-2a lysate, murine neuroblastoma) (b)

Fig. 7

ISAP induced expansion of T-reg cells: mice were vaccinated twice, 1 week apart with empty MP or MP containing CpG and TL (ISAP) and then sacrificed 1 week later for splenocyte harvest. The compartment of CD4 + CD25 + Foxp3 + Treg cells was determined by flow cytometry following 5 days of splenocyte culture with irradiated tumor cells. The upper panel shows CD4 + CD25 + cells as a percent of splenocytes, whereas the lower panel shows the FoxP3 + CD25 + cells as a percent of CD4 + cells. When empty microparticles were used for vaccination, only 3% of CD4 + T-cells expressed the markers of phenotypic T-reg. However, following vaccination with the ISAPs, 19.4% of the CD4 + T-cells were positive for the T-reg markers. Cyclophosphamide treatment appeared to suppress the ISAP induced compartment of T-reg in which only 2.2% of CD4 + cells subsequently expressed FoxP3 and CD25

Fig. 8

CD25 antibody effectively suppressed the T-reg compartment. Anti-CD25 antibody was administered 24 h prior to vaccination with ISAP. One week later, splenocytes were harvested from the mice receiving either ISAP alone or ISAP plus anti-CD25 Ab. Splenocytes were cultured with irradiated tumor cells for 5 days following which flow cytometry was performed to define the T-reg compartment. The upper panel shows CD4 + CD25 + cells as a percent of splenocytes, whereas the lower panel shows the FoxP3 + CD25 + cells as a percent of CD4+ cells. Anti CD25 effectively suppressed the CD4 + CD25 + and the FoxP3 + CD25 + compartment following ISAP vaccination

Fig. 9

Prophylactic tumor model. Mice were vaccinated twice, 1 week apart and then a week later with various preparations of MPs and then 7 days later challenged with high dose live B16 melanoma (5 × 10⁵). Remarkably, mice were protected from developing tumor only when tumor lysate and CpG ODN were co-loaded into the microparticles (ISAPs) and anti-CD25 antibody (@-CD25) was used to suppress the T-reg compartment. Low dose cyclophosphamide (CY) was more protective than vaccine alone, but failed to induce protection to the same level observed for the anti-CD25 Ab. Complete protection was achieved in 75% of mice for 6 weeks and in 63% of mice indefinitely when the anti-CD25 Ab was combined with ISAP vaccination (n = 8)