Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response

Abstract

T helper type 2 (T(H)2)-mediated immune responses are induced after infection with multicellular parasites and can be triggered by a variety of allergens. The mechanisms of induction and the antigen-presenting cells involved in the activation of T(H)2 responses remain poorly defined, and the innate immune sensing pathways activated by parasites and allergens are largely unknown. Basophils are required for the in vivo induction of T(H)2 responses by protease allergens. Here we show that basophils also function as antigen-presenting cells. We show that although dendritic cells were dispensable for allergen-induced activation of T(H)2 responses in vitro and in vivo, antigen presentation by basophils was necessary and sufficient for this. Thus, basophils function as antigen-presenting cells for T(H)2 differentiation in response to protease allergens.

Figure 1. Basophils, but not dendritic cells, are necessary for T_H2 differentiation in vitro

(a) Splenic CD4⁺ cells from DO11.10 x 4get mice were co-cultured with OVAp and indicated ratios of MACS purified BMBs and BMDCs from Balb/c mice. The percentage of IL-4-eGFP⁺, out of total CD4⁺ cells, was measured after three days. (b) CD4⁺ cells as in (a) were mixed with DCs from the indicated sites in papain immunized C3H.T4d mice (draining = popliteal lymph node and non draining = brachial lymph node), in the presence (black) or absence (white) of BMBs. (c) Proliferation of CFSE labeled CD4⁺ cells after co-culture with BMBs. Percentage is of proliferating cells out of total live CD4⁺ cells. (d) Splenic CD4⁺ as in (a) were mixed with unstimulated (white bar) or papain stimulated (black bar) BMBs. (e) T_H2 differentiation in the presence of blocking antibodies to MHC class II. (f) T_H2 differentiation in cultures with FACS purified DO11.10 x 4get CD4⁺MHCII^- cells and FACS purified BALB/c BMBs. Unless noted, BMBs were stimulated with papain prior to co-culture. p values were calculated using the Student’s t test, comparing conditions with or without basophils or with or without papain stimulation. *, p<0.01; **, p<0.001; ***, p<0.0001. Data shown are representative of at least three independent experiments.

Figure 2. Basophil mediated T_H2 differentiation in vitro is dependent upon IL-4 and TSLP

Unstimulated (white bars) or papain stimulated (black bars) BMBs from IL-4 sufficient (BALB/c) or deficient (Il4^−/−) mice were co-cultured with splenic CD4⁺ cells from DO11.10 x 4get mice and OVAp. T_H2 differentiation was assessed by the percentage of IL-4-eGFP⁺ CD4⁺ T cells after 3 days of co-culture. p values were calculated using the Student’s t test, comparing Il4^−/− conditions to corresponding BALB/c conditions. *, p<0.01; **, p<0.001; ***, p<0.0001. Data shown are representative of five experiments.

Figure 3. Basophils express and upregulate MHC Class II after papain stimulation and are capable of forming synapses with T cells in vitro

(a) Q-PCR expression of Ciita in BMBs activated for 4 hours in vitro as indicated compared to expression in various other hematopoetic cells. Mast cell, macrophage and DC RNA was isolated from bone marrow derived cell cultures. T cell RNA was derived from FACS sorted splenic CD3⁺CD4⁺ cells. Q-PCR expression of MHC Class II and related genes in BMBs from BALB/c mice activated for 4 hours as indicated (b). (c) FACS staining of lymph node basophils (black histogram) versus peripheral blood basophils (gray shaded histogram) three days after papain immunization. Dashed histogram in I-A—I-E stain indicates isotype control. (d) Mast cell depleted cultures of BMBs were stimulated with inactive or active papain. I-A—I-E expression is shown on live basophils cells after stimulation. Percentages shown are of the gated population out of live basophils. (e) Immunofluorescence of MHC Class II expression in BMBs after stimulation with inactive or active papain. MHC Class II (red), DAPI (blue); 20x magnification. (f) Immune synapse formation 60 minutes after co-culture of papain activated BMBs (BALB/c) and splenic CD4⁺ T cells (DO11.10). TCRβ (green), MHC Class II (red); 100x magnification. Data shown are representative of at least four independent experiments. ND indicates no transcript detected.

Figure 4. Basophils are capable of endocytosis of soluble, but not particulate antigens

(a) Endocytosis of soluble OVA-FITC after 3 hour culture by B cells, BMDCs or BMBs in vitro at 37°C (black lined histogram) or at 4°C (gray shaded histogram). (b) Endocytosis of soluble proteins by basophils leads to functional antigen presentation as measured by T_H2 differentiation. Unstimulated (white bars) or papain stimulated (black bars) BMBs were co-cultured with splenic CD4⁺ T cells from DO11.10 x 4get mice in the presence or absence of OVA protein. T_H2 differentiation is calculated as a measure of IL-4-eGFP⁺ CD4⁺ T cells three days after co-culture. (c) Phagocytosis of fluorescently labeled 2μm latex beads after overnight culture by B cells, BMDCs or BMBs in vitro at 37°C (black lined histogram) or at 4°C (gray shaded histogram). p values were calculated using the Student’s t test, comparing conditions with or without OVA protein. *, p<0.01; **, p<0.001. Data shown are representative of at least three independent experiments.

Figure 5. Migratory DCs are not necessary for basophil migration and T_H2 differentiation after papain immunization

4get mice were immunized with 50μg of active papain in 10μl of PBS in the distal pinna. The injection site was either removed or left intact 2 hours after immunization. Basophil migration (a) and T_H2 differentiation (b) in the ipsilateral cervical lymph node was assessed 3 and 4 days later, respectively. Percentages shown are that of the gated population out of total live cells from the cervical lymph node. FACS plots and percentages shown are representative of three independent experiments.

Figure 6. DCs and DC derived MHC Class II is not required for T_H2 differentiation after papain immunization

DC depletion after DT injection of CD11c-DTR-GFP → BALB/c bone marrow chimeras (a). (b) Basophil migration in chimeras after OVA immunization without DT depletion (white bar) or after OVA & Papain immunization without (gray bar) or with (black bar) DT depletion of DCs. Values are percentage of live cells. (c) T cell differentiation (based on cytokine production after in vitro restimulation) in chimeras after immunizations with (black bars) or without (white bars) DT injection. Chimeras first received CD4⁺ splenic T cells from DO11.10 mice and were immunized as indicated. (d) Basophil migration in CD11c-IABB mice after OVA (white bar) or OVA & papain (black bar) immunization. (e) T cell differentiation, assessed as in (c), after OVA & papain or OVA & LPS immunization in CD11c-IABB (IABB) mice (black bars) or BL/6 mice (white bars). OT-II CD4⁺ T cells were transferred into CD11c-IABB mice, which were subsequently immunized as indicated. p values were calculated using the Student’s t test, comparing conditions to No DT injection (b & c), OVA immunization (d) or to wild type mice (e). *, p<0.01; **, p<0.001; ***, p<0.0001. ND indicates none detected. Data shown are representative of at least three independent experiments.

Figure 7. Antigen presentation by basophils is sufficient for T_H2 differentiation

T_H2 differentiation as measured by T cell IL-4 production on in vitro restimulation of CD4⁺ T cells isolated four days after transfer of antigen coated (white bars) or uncoated (black bars) BMBs into BL/6 or CIITA ko mice. p values were calculated using the Student’s t test, comparing antigen loaded conditions to no peptide conditions. *, p<0.01; **, p<0.001. Data shown are representative of three independent experiments.