The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance

Abstract

We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)-associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80(-/-) mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80(-/-) APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80(-/-) mice generated an efferent CD8(+) T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80(-/-) mice by adoptive transfer of F4/80(+) APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8(+) T reg cells.

Figure 1.

Gene targeting of the mouse F4/80 gene. (A) Partial restriction maps of the mouse F4/80 gene, the targeting construct, and the resulting targeted allele. The β-galactosidase and pGK-Neo gene cassettes are shown as black and white boxes, respectively. White boxes with numbers indicate exons. The 5′-UTR is represented by a stippled box. DNA probes A and B used for Southern blot analysis are shown. The expected HindIII fragments of the WT (10.2 kb) and the targeted alleles (14.0 kb) detected by probes A and B are also shown. Arrows with numbers indicate the positions and orientations of the primers used for the PCR genotyping reaction. (B) Southern blot and PCR genotyping. Tail DNA was digested with HindIII and hybridized with probe A (top) and probe B (middle). The same genomic DNA was analyzed using primers 1, 2, and 3. (C) Northern blot hybridization and (D) RT-PCR analysis of total RNA from thymus of F4/80^+/+, +/−, and −/− animals using (C) the full-length F4/80 cDNA probe and (D) primers specific for the F4/80 (top) and Hprt (bottom) cDNAs. (E) mAb F4/80 staining in the spleens and livers (Kupffer's cells, arrow) of the F4/80^+/+ and −/− animals. Magnification at 200 (spleen), 400 (liver).

Figure 2.

Immunohistochemical analysis of the F4/80^−/− macrophage. mAb 3D6 (anti-sialoadhesin) staining in the (A, B) livers and (C, D) and spleens of the (A, C) F4/80^+/+ and (B, D) −/− animals. 3D6⁺ Kupffer's cells and macrophages (brown) were found in the livers and the red pulp and marginal zone areas of spleens of both (A, C) F4/80^+/+ and (B, D) −/− mice. The strongest 3D6 staining is on macrophages in the inner region of the marginal zone (marginal metallophils), forming a tight ring (arrow) around the white pulp (C, D). Magnification at 400 (A, B), 200 (C, D). (E, F) mAb 5C6 staining in the spleens and (G, H) mAb FA-11 staining in the livers of the (E, G) F4/80^+/+ and (F, H) −/− animals. mAb FA-11 is specific for macrosialin (mouse CD68), whereas mAb 5C6 specifically recognizes type 3 complement receptor on myeloid cells. Magnification at 200 (E, F), 400 (G, H).

Figure 3.

The role of F4/80 in the generation of regulatory T cells in vivo after a.c. inoculation of antigen. Enriched splenic T cells were collected from WT and F4/80^−/− mice that had received a.c. OVA 7 d earlier and were mixed with WT, OVA-pulsed PECs, and OVA-primed T cells. The cell inoculum was given intradermally to the pinnae of naive B6 mice. Ear thickness was determined before and at 24 and 48 h after injection. Data are represented as mean change in ear thickness at 24 h after injection ± SEM. The composition of the cell inoculum is indicated under each bar in the graph. *, P < 0.01; n = 5/group.

Figure 4.

The role of F4/80 in the generation of regulatory T cells in vitro. (A) Assessment of F4/80⁺ cells in the circulation after a.c. or i.v. inoculation of antigen. The bar graph represents the number of F4/80⁺ cells per million blood mononuclear cells in naive mice and 3 d after a.c. or i.v. inoculation of OVA. The number of F4/80⁺ cells was determined from four pooled, splenectomized mice for the a.c. and i.v. groups and five mice from a control group. The data shown are representative of two experiments in which similar results were obtained. (B) Generation of T reg cells with F4/80^−/− APC in the presence or absence of anti F4/80 mAb. The bar graph shows the magnitude of ear swelling (ordinate) as a measure of DTH responses generated in naive B6 mice. Mice were given an intradermal ear injection of a cell mixture containing OVA-primed spleen cells and T reg cells. T reg cells were generated by coculturing naive spleen cells for 5 d with WT or F4/80^−/− PECs that had been treated overnight with OVA with or without TGFβ2. Before the addition of the naive spleen cells, cells were cultured in presence or absence of anti-F4/80 mAb. Ear thickness was determined before ear pinnae injection and at 24 and 48 h after injection. *, P < 0.01; n = 5/group.

Figure 5.

The role of F4/80 in the generation of regulatory T cells in vivo after low-dose oral gavage of antigen. (A) Low-dose oral tolerance experiments in WT and F4/80^−/− mice: the ear swelling DTH response to OVA of F4/80^−/− mice immunized with OVA in CFA s.c. and ear challenged with OVA intradermally in the ear pinnae (positive DTH) versus those fed OVA (gavage). The treatment of the mice is indicated below the abscissa under each bar. Naive F4/80^−/− mice that received only OVA by ear challenge served as the negative DTH control. The number (n) and treatment of the mice are indicated along the abscissa under each bar graph. The change in ear thickness is listed on the ordinate. (B) Low-dose antigen gavage generated an efferent T reg cell in WT but not in F4/80^−/−mice. 7 d after OVA-gavage, enriched T cells from spleens were mixed with primed T cells (effector) and OVA-pulsed APCs (stimulator) before being injected into the ear pinnae of naive mice. Phenotype of the cells in the mixture injected is indicated under the abscissa. The change in ear swelling is indicated on the ordinate. *, P ≤ 0.05. (C) LAT assay for efferent CD8⁺ T reg cells in WT mice that received antigen by gavage. 7 d after OVA-gavage, T cells were enriched from spleens and were either depleted of CD4⁺ T cells (GK1.5 and complement), depleted of CD8⁺ T cells (2.43 and complement), or kept unmanipulated. The enriched T cells were mixed with primed T cells (effector) and OVA-pulsed APCs (stimulator) before being injected into the ear pinnae of naive mice. The phenotype of the cells in the mixture injected is indicated under the abscissa. The change in ear swelling is indicated on the ordinate. *, P ≤ 0.05. Depletion of cells was confirmed using flow cytometry (not depicted).

Figure 6.

Reconstitution of F4/80^−/− mice with F4/80⁺ APC restores peripheral tolerance. (A) To demonstrate antigen specificity of efferent suppression in two models of tolerance where animals received OVA either by intracameral inoculation or gavage before challenge in the ear with OVA or human serum albumin (HSA) (unrelated antigen). The change in ear swelling is on the ordinate, and the antigen regimen is displayed below bars under the abscissa. (B) In the ACAID tolerance model, F4/80^−/− mice were reconstituted with 10⁶ F4/80⁺ bone marrow–derived APCs (BMAPCs) 1 d before inducing ACAID with OVA a.c. (C) In the low-dose oral tolerance model, F4/80^−/− mice were reconstituted with 10⁶ F4/80⁺ BMAPCs . The experiment was repeated twice with similar results. (D) F4/80^−/− mice were reconstituted with 10⁶ F4/80⁺ adherent spleen cells. The next day, mice were given OVA gavage, and 7 d later they were immunized with OVA s.c. After another week, they were challenged intradermally in the ear. DTH responses were measured as an increase in ear thickness. The treatment of the mice is indicated below the abscissa under each bar. The change in ear thickness is indicated on the ordinate. Significant (P ≤ 0.05) differences between groups are indicated with an asterisk.