Recruitment of bone marrow CD11b+Gr-1+ cells by polymeric nanoparticles for antigen cross-presentation

Abstract

The objective of this study was to investigate the function of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on the activation of antigen-specific CD8⁺ T cell responses via the CD11b⁺Gr^-1⁺ myeloid subpopulations in murine bone marrow (BM). PLGA NPs containing ovalbumin (OVA) were fabricated by the double-emulsion method. The CD11b⁺Gr-1^lowLy-6C^high and CD11b⁺Gr-1^highLy-6C^low subsets from mice bone marrow were sorted and treated with the PLGA/OVA NPs, followed by co-culture with the carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-I CD8⁺ cells. Co-culture of OT-I CD8⁺ T cells with PLGA/OVA NPs-primed CD11b⁺Gr-1⁺ subsets upregulated the expression of IL-2, TNF-α, INF-γ, granzyme B, and perforin, resulting in proliferation of CD8⁺ T cells and differentiation into effector cytotoxic T lymphocytes (CTLs). In vivo proliferation of CFSE-labelled OT-I CD8⁺ cells in response to OVA was also obtained in the animals immunized with PLGA/OVA NPs. The results presented in this study demonstrate the ability of polymeric NPs to recruit two CD11b⁺Gr^-1⁺ myeloid subsets for effective presentation of exogenous antigen to OT-I CD8⁺ T cells in the context of major histocompatibility complex (MHC) class I, leading to an induction of antigen-specific cell proliferation and differentiation into effector cells.

Figure 1

Scanning microscopic examination of PLGA/OVA NPs and sorting of the Gr-1⁺ subsets from mouse bone marrow (a), SEM image of the PLGA/OVA NPs. Scale bar = 1 μm. (b) Size distribution of the PLGA/OVA NPs. (c), Illustration of the gating scheme of Gr-1^high and Gr-1^low subsets from mouse bone marrow. Bone marrow cells were first gated on single live cells and sorted by flow cytometry based on their expression of Lin2 (CD3/CD19/B220/Ter119/NK1.1), CD11b, Ly-6C, and Gr-1. (d,e) Dot plots displaying the heterogeneity of the Gr-1^low subset that partly express CD115, whereas Gr-1^high cells do not. Figures (f,g) illustrate the microscopic morphology of the CD11b⁺Gr-1^high (f) and CD11b⁺Gr-1^low (g) subsets after staining with the Wright-Giemsa stain. Scale bar = 10 μm.

Figure 2. PLGA/OVA NPs primed Gr-1^high and Gr-1^low subsets from the murine bone marrow by the PLGA/OVA NPs induced cross-presentation of antigens in OT-I CD8⁺ T-cells.

The representative flow cytometric plots, illustrating (a), the gating scheme of OT-I TCRVβ5⁺CD8⁺ T-cells after co-culture with PBS, OVA, PLGA NPs, or PLGA/OVA NPs -treated Gr-1⁺ subset and (b,c), progressive cell division of CFSE-labelled OT-I CD8⁺ T cells in response to NPs treatment. Proliferation and the percentages of CD25⁺ and CD69⁺ T cells after the treatment are shown in (d,e); (f) illustrates the delta mean fluorescence intensities (ΔMFI) of CD25 and CD69 in the gated OT-I CD8⁺ T cells, which are presented as the mean values of four separate experiments relative to the isotype controls. Asterisks denote statistically significant differences vs. the PBS control in the same group (*p < 0.05; **p < 0.01; ***p < 0.001; by one-way ANOVA).

Figure 3. Time-course of surface expression of Ly-6C, CD115, CD11c, F4/80, and I-A/I-E in the Gr-1^high and Gr-1^low subsets after treatment with the PLGA/OVA NPs.

(a) Gr-1^high and (b), Gr-1^low cells were treated with PLGA/OVA NPs in complete RPMI 1640 medium containing 5 ng/ml GM-CSF, and incubated at 37 °C for 0, 8, 16, and 24 hrs, followed by flow cytometric analysis. (c) The expression of I-A/I-E and CD115 in the DP (CD11c⁺F4/80⁺) and DN (CD11c¯F4/80¯) populations at 24 hrs after treatment of Gr-1^low cells with the PLGA/OVA NPs. (d) Visual examination of the Gr-1^low cells after treatment with the PLGA/OVA NPs with a phase contrast microscope and a digital camera. The presence of PLGA/OVA NPs can still be observed at 8 hrs after incubation (arrow head), but these NPs were cleared up at 16 hrs (arrow head) and extensively ingested at 24 hrs post-incubation when the spindle-shaped cells became abundant.

Figure 4. PLGA/OVA NPs-treated CD11b⁺Gr-1⁺ cells efficiently induced the expression of IL-2, TNF-α, and IFN-γ in the OT-I CD8⁺ T cells, as determined by intracellular cytokine (ICC) staining.

Intracellular cytokine and surface staining by flow cytometric analysis in the PLGA/OVA NPs-treated CD11b⁺Gr-1⁺ cells, showed the enhanced expression of (a), IL-2, (b), TNF-α, and (c), IFN-γ. The grey and tinted area represents the isotypes. (d,e) Statistical analysis of the percentages of cytokine-expressing T cells and the delta mean fluorescence intensities (ΔMFI) of OT-I CD8⁺ T cells relative to the isotype controls, performed by one-way ANOVA. Data are displayed as the mean ± standard error mean (SEM) from five separate experiments. Asterisks indicate statistically significant differences vs. the PBS control in the same group (*p < 0.05; ***p < 0.001; by one-way ANOVA).

Figure 5. PLGA/OVA NPs-treated Gr-1^high or Gr-1^low subsets induced the expression of perforin and granzyme B in the OT-I CD8⁺TCRVβ5⁺ T cells, resulting in the cytotoxic lymphocyte (CTL) effect.

Intracellular and surface staining and analysis by flow cytometry shows the expression of (a) perforin and (b) granzyme B in the OT-I CD8⁺TCRVβ5⁺ T cells, after co-culture with PBS, OVA, PLGA NPs, or PLGA/OVA NPs-treated Gr-1^high or Gr-1^low subsets. Grey and tinted area are isotype controls. (c), The data, presented as the mean ± standard error mean (SEM) of five separate experiments, showed the increased percentages of perforin and granzyme B-expressing cells, relative to the isotype controls, in the OT-I CD8⁺ population after co-culture with the CD11b⁺Gr-1⁺ subsets, pre-treated with PBS, OVA, PLGA NPs, or PLGA/OVA NPs. Asterisks denote statistically significant differences vs. the PBS control in the same group (*p < 0.05; ***p < 0.001; by one-way ANOVA). (d,e) Fluorescence microscopic examination of CD8α⁺TCRVα2⁺ T cells, after co-culture with PBS or PLGA/OVA NPs-treated Gr-1^high or Gr-1^low subsets, confirmed the expression of perforin (d) and granzyme B (e). (f) PLGA/OVA NPs-primed Gr-1⁺ cells induced cytotoxic T-lymphocyte (CTL) activity of the OT-I CD8⁺ T cells. Lysis of the target cells was determined using the LDH release assay. The values presented are the mean values ± SEM of triplicate experiments. Asterisks indicate statistically significant differences between the SIINFEKL-pulsed EL4 cells and the untreated controls at the same E:T ratio (**p < 0.01; ***p < 0.001; by t-test).

Figure 6. Immunization of mice with PLGA/OVA NPs increased the titres of anti-OVA antibody and induced proliferation of OT-I CD8⁺ T cells in vivo.

(a) Antibody titres are expressed as the dilution factor giving OD 450 values above 0.1. The data represent mean titres ± SEM of (a), IgG1, (b) IgG2a, and (c), IgG2b from three independent experiments. Statistically significant differences between the PLGA/OVA NPs-treated group vs. the OVA-control were denoted with asterisks (*p < 0.05; **p < 0.01; ***p < 0.001; by t-test). (d) The proliferation of adoptively transferred OT-I CD3⁺CD8⁺TCRβ5⁺ cells, isolated from spleen and lymph nodes of the recipient mice that were pre-treated four days earlier with PBS, OVA, PLGA NPs, or PLGA/OVA NPs, was analysed by flow cytometry. The gating in the graphs shows the percentage of the proliferating OT-I CD8⁺ cells. Data presented are representative of three independent experiments.