Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17 T-cell differentiation

Abstract

Neutrophils play a major role in the innate immune system and are normally considered to be short-lived effector cells that exert anti-microbial activity and sometimes immunopathology. Here, we show that these cells possess an additional function as professional antigen-presenting cells capable of priming a T(h)1- and T(h)17-acquired immune response. Using flow cytometry, fluorescence microscopy and western blotting, we show that mouse neutrophils express MHC class II and co-stimulatory molecules CD80 and CD86 after T-cell co-incubation. Neutrophils pulsed with ovalbumin (OVA) process and present peptide antigen to OVA-specific T cells in an MHC class II-dependent manner. Importantly, we demonstrate that neutrophils can prime antigen-specific T(h)1 and T(h)17 immune responses even without the addition of exogenous cytokines to cell cultures.

Fig. 1.

Source of neutrophils and absence of APC populations in purified neutrophils. (A) Expression of Ly6G by thioglycollate-elicited neutrophils isolated by centrifugation over Percoll. The numbers indicate the relative percentage of cells falling within each indicated gate. PMN were sorted into Ly6G high (B) and intermediate (C) expressing populations. Insets in panels B and C show the morphological appearance of the flow-sorted populations. There was minimal cross contamination as determined by staining for DC (CD11c, panel D), macrophages (F4/80, panel E) and B cells (CD19, panel F). Data are representative of at least three independent experiments.

Fig. 2.

Co-incubation with T cells induces expression of MHC class II and co-stimulatory molecules on neutrophils. (A and C) Expression of MHC class II molecules on Ly6C/G (Gr-1)-gated neutrophils without (A) or with (C) 2-h T-cell co-incubation (red histograms). Gray shaded histograms show isotype antibody staining. (B and D) Neutrophils were imaged by fluorescence microscopy after staining with anti-Ly6C/G antibody (green), anti-MHC class II antibody (red). Nuclei were stained with DAPI (blue). MHC class II expression on neutrophils without (B) and with (D) T-cell co-incubation is shown. (E and F) Expression of CD86 (E) and CD80 (F) on Ly6C/G-gated neutrophils without T-cell co-incubation (shaded histogram) relative to expression after T-cell co-incubation (red histogram). The insets in panel E and F show isotype antibody staining (shaded blue histogram) compared with CD86 and CD80 expression on neutrophils with T-cell co-incubation respectively (red histogram). MHC class II and co-stimulatory molecule flow cytometry data are representative of at least four independent experiments. The microscopy data are representative of two independent experiments.

Fig. 3.

Biochemical evidence for MHC class II expression on neutrophils. (A) Neutrophils and T cells were co-cultured for 2 h at 37°C at a ratio of 1:10, then cells were labeled with Ly6G-specific antibody. Expression of Ly6G before (A, 10% positive) and after (B, 98% positive) cell sorting for neutrophils. (C) Western blot showing MHC class IIβ expression on neutrophils after T-cell co-incubation (Post-T). C also shows GAPDH loading control blotting from the same experiment. Contr, neutrophil lysate prepared without prior T-cell incubation. These data were repeated twice with the same result.

Fig. 4.

Physical contact between neutrophils and T cells is required for up-regulation of MHC class II by neutrophils. (A) Background expression of cell surface MHC class II molecules on Ly6C/G-positive neutrophils prior to incubation with T cells. (B) Expression of MHC class II molecules on the surface of Ly6C/G-positive neutrophils after 2-h co-incubation with T cells. (C) Expression of MHC class II molecules by neutrophils when the T cells and neutrophils are separated by a Transwell membrane. The data are representative of three independent experiments.

Fig. 5.

Neutrophils process and present antigen to stimulate T-cell proliferation. (A) Neutrophils were pre-incubated with OVA, then cells were added to OVA-specific OT-ll cells at a PMN to T cell ratio of 1: 10. Four days later, cells were examined under the microscope. The arrow points to one of three neutrophils in this image. In panels B–E, the ability of Percoll gradient-isolated neutrophils (97% purity) to stimulate immunomagnetic bead-isolated CD4⁺ T cell (98% purity) proliferation was examined. (B) Non-pulsed neutrophils induce low levels of OT-II T-cell proliferation in day 4 cultures as measured by CFSE dilution of labeled CD4⁺ T cells. (C) Proliferation of OVA-specific OT-II T cells after day 4 co-culture with neutrophils pre-incubated (4 h, 37°C) with whole OVA. (D) OT-II T cells proliferation after 4-day culture with OVA peptide-pulsed PMN. (E) Addition of anti-MHC class II blocking antibody (M5/114) prevents OT-II proliferation stimulated by OVA peptide-pulsed neutrophils. Insets in B through E show CFSE peak dilution histograms. Samples in B through E were incubated at a ratio of 10 T-cells for every one neutrophil. In panels F–I, the experiments were reiterated using flow sorted Ly6G^high neutrophils and CD4⁺ OT-II T cells. (F) Neutrophil purity after cell sorting (99% Ly6G positive). (G) Purity of OT-II T cells following cell sorting (98% CD4 positive). (H) Day 4 proliferation of sorted CFSE-labeled OT-II CD4⁺ T cells after incubation with sorted neutrophils pulsed with OVA peptide. (I) Proliferation of sorted OT-II T cells after incubation with non-pulsed sorted neutrophils. (J) Proliferation of OVA-specific OT-II T cells following incubation with OVA peptide-pulsed neutrophils from MHC class II expressing C57BL/6 mice. (K) Proliferation of CD4⁺ T cell following 4-day incubation with OVA peptide-pulsed neutrophils from MHC class II-deficient mice. Neutrophil purity, based on Ly6G expression, is shown in the inserts and was 96.4 and 96.6% in J and K, respectively. Samples in (F) through (K) were incubated at a ratio of five T-cells for every one neutrophil. Experiments in (A–D) were performed five times with the same result and the MHC blocking experiment (E) was performed three times with the same result. The cell sorting experiment (F–I) was performed three times and experiments with MHC class II KO cells were performed two times with the same result.

Fig. 6.

OTII CD4⁺ T-cells exhibit a naive phenotype prior to antigen presentation by neutrophils. Expression of CD44 and CD62L by CD4⁺ T-cells from a naive spleen of an OTII mouse (A) and expression of these markers after neutrophils antigen presentation in (B). Expression of CD25 on CD4⁺ T cells before and after neutrophils antigen presentation is shown in (C and D), respectively. The numbers indicate the relative percentage of cells falling within each indicated gate.

Fig. 7.

OVA-pulsed neutrophils drive differentiation of T_h1 and T_h17 cells in vitro. (A–C) PMN and bone marrow-derived DC were pulsed with OVA peptide or medium alone (Med) then cultured with OVA-specific T cells. At day 4 after culture initiation, supernatants were collected and assayed for IFN-γ (A), IL-17 (B) and IL-4 (C). In (D and E) similar cultures were initiated using neutrophils from wild-type and MHC class II knockout mice. Day 4 supernatants were tested for IFN-γ (D) and IL-17 (E, where * indicates P <0.001). In panels (F and G), the co-cultures were subjected to intracellular cytokine staining following incubation with non-pulsed (F) and OVA peptide-pulsed (G) neutrophils. The results in F and G show intracellular IFN-γ and IL-17 after gating on CD4⁺ T cells. Panel (H) shows intracellular staining on CD4-negative gated population, while panel (I) shows the total number of cells staining positive for IL-17, IFN-γ or both (DP, double positive) in both the CD4 negative and positive populations. These experiments were performed twice with the same result.

Fig. 8.

T_h17 induction by neutrophils is both IL-6 and TGF-β independent. Purified neutrophils (A) and bone marrow-derived DC (B) were pulsed with OVA peptide and incubated with OVA-specific CD4⁺ T cells for 4 days in the presence of a T_h17 skewing cocktail. In the indicated samples, an anti-skew mAb cocktail was included. On day 4, cultures were spun down and fresh medium supplemented with IL-2 added to co-cultures. Supernatant was collected 3 days after fresh media addition. Cytokine IL-17 levels were measured by ELISA. M, medium only; Skew, IL-17 skewing cocktail consisting of IL-6 (20 ng ml⁻¹), TGF-β (1 ng ml⁻¹), anti-IFN-γ (10 μg ml⁻¹) and anti-IL-12 (10 μg ml⁻¹); αSkew, anti-skewing cocktail consisting of anti-IL6 and anti- TGF-β mAb each at 10 μg ml⁻¹.