Thymus-blood protein interactions are highly effective in negative selection and regulatory T cell induction

Abstract

Using hen egg-white lysozyme, the effect of blood proteins on CD4 thymic cells was examined. A small fraction of i.v. injected hen egg-white lysozyme rapidly entered the thymus into the medulla. There it was captured and presented by dendritic cells (DCs) to thymocytes from two TCR transgenic mice, one directed to a dominant peptide and a second to a poorly displayed peptide, both presented by MHC class II molecules I-A(k). Presentation by DC led to negative selection and induction of regulatory T cells, independent of epithelial cells. Presentation took place at very low levels, less than 100 peptide-MHC complexes per DC. Such low levels could induce negative selection, but even lower levels could induce regulatory T cells. The anatomy of the thymus-blood barrier, the highly efficient presentation by DC, together with the high sensitivity of thymic T cells to peptide-MHC complexes, results in blood protein Ags having a profound effect on thymic T cells.

Figure 1. Limiting amounts of injected HEL enters the thymus and localizes to the medulla

Protein bound radioactivity is shown in (A) the thymus and spleen and in (B) blood at different times post-injection with 45.4 μg ¹²⁵I-HEL. Data is presented as % input dose recovered from the thymus and spleen and B, in the trichloroacetic precipitated fraction from serum. There are 5-6 mice in each group. Thymus, (*) indicates p< 0.05, (**) p=0.0087. Spleen, (**) indicates p=0.0043. Serum, (**) indicates p=0.0043. (C-G) Thymic sections from a B10.BR mice injected with 10mg purified HEL (C-E) or 1 mg HEL biotin (F-G). Mice were sacrificed at 20 min and 15 min respectively. In panels C-E, 6 μm sections were stained using monoclonal antibodies for (C) HEL (green) and (D) CD11c (red). Panels (E) shows overlay of HEL and CD11c staining. Inset in panel C shows region indicated by arrow. Panel (F) shows streptavidin staining of a thymic section from a B10.BR mouse injected with 1 mg HEL-biotin or (G) an uninjected B10.BR mouse.

Figure 2. Negative selection and Foxp3 expression in LB11.3 and 3A9 mice injected with HEL

(A) top. Representative CD4 and CD8 flow cytometry profiles of thymocytes from 3A9 and LB11.3 TCR transgenic mice injected with saline (PFS), 5, or 50 μg of HEL. Bottom shows corresponding Foxp3 expression gated on CD4SP. (B) Total number of thymocytes within CD4SP/DP from 3A9 mice injected with the indicated amounts of HEL. IG12 indicates thymocytes positive for a monoclonal antibody to the TCR of 3A9. Bars correspond to pooled data from 15 mice (PFS), 7 mice (10ng), 13 mice (500 ng), 11 mice (1μg), 6 mice (5μg), 4 mice (50μg) and 3 mice (1000μg). (C) Total number of thymocytes within CD4SP/DP sub-population from LB11.3 mice injected with titrated amounts of HEL. Bars represent pooled data from 17 mice (PFS), 13 mice (5μg), 15 mice (10μg), 4 mice (30μg), 20 mice (50μg) and 4 mice (100 μg) (D) Total number of Foxp3⁺CD4SP thymocytes in 3A9 mice shown in (B). (*) indicates p=0.0148. (E) Total number of Foxp3⁺CD4SP thymocytes in LB11.3 mice shown in (C). Each symbol represents an individual mouse. (F) Foxp3 expression within 1G12^hi and 1G12^lo CD4SP in 3A9 mice shown in figure D. (G) Foxp3 expression within different Vα2 expressing CD4SP subset in LB11.3 mice shown in figure E. Unless exact p values are provided, for all figures, (****) indicates p<0.0001, (***) indicates p<0.001, (**) indicates p<0.01 and (*) indicates p<0.05. All graphs are based on flow cytometry data gating on CD4SP cells.

Figure 3. Role of TCR alpha chain gene rearrangement in Foxp3⁺CD4SP Treg induction in mice injected with HEL

(A) Total number of CD4SP and DP in 3A9 alpha^-/- injected with HEL. Data are pooled from two independent experiments with individual bars corresponding to 8 mice (PFS), 10 mice (0.5μg), 12 mice (1μg) and 4 mice (5μg). (B) Total number of CD4SP and DP from LB11.3 alpha^-/- mice injected with HEL. Data are pooled from five independent experiments with individual bars corresponding to 17 mice (PFS), 9 mice (0.5μg), 9 mice (1μg), 10 mice (2.5μg), 17 mice (5μg), 15 mice (10μg), and 11 mice (50μg). (C) Total number of Foxp3⁺CD4SP cells from 3A9 alpha^-/- mice shown in (A). (D) Total number of Foxp3⁺CD4SP cells from LB11.3 alpha^-/-shown in (B). All graphs are derived from flow cytometry data gating on CD4SP. Where indicated, (*) denotes p<0.05 and (**) denotes p<0.01.

Figure 4. Presentation of blood-borne HEL by thymic DC and not TECs

(A) Presentation of HEL by CD11c ⁺ DC isolated at 30 min and 36 h from B10.BR mice injected with 50μg HEL. (B) Corresponding presentation by 2-3×10⁴ TECs isolated from the same mice and cultured as in (A). For panels A-B, APCs were cultured for 24 h with 5×10⁴ 3A9 T-cell hybridoma and IL-2 production was measured by CTLL proliferation indicated by ³H-Thymidine incorporation. Proliferation of primary (C) 3A9α^-/- and (D) LB11.3 T-cells in response to presentation by CD11c⁺ DC and CD45^- TECs from mice injected with 50μg HEL. (E-F) Proliferation of primary 3A9 T-cells in response to presentation by (E) CD11c⁺ DC or (E, F). Sorted CD45^-EpCAM⁺BP-1^- mTECs isolated from mice injected with 1mg HEL. For primary proliferation, APCs were co-cultured with T-cells for 4 days and ³H-Thymidine incorporation was measured. In Panels C-F, mice were sacrificed 30 min post-injection and APCs isolated from un-injected B10.BR mice were used as controls.

Figure 5. HEL is efficiently presented by conventional thymic DC

HEL presentation by dendritic cell subsets isolated 30 min post-injection and sorted based on differential expression of CD11c, CD45RA, Sirpα, and CD8α. APC were cultured with (A) 3A9 T-cell hybridoma or (B) primary 3A9 T-cells. (C) HEL Presentation to 3A9 T-cell hybridoma by sorted CD8α⁺ and (D) CD8α^- CD11c^hi dendritic cells isolated 30 min and 36 h post-injection. For hybridoma assays, APCs were cultured for 24 h with 3A9 T-cell hybridoma and IL-2 production was measured indirectly by CTLL proliferation indicated by ³H-Thymidine incorporation. For primary proliferation, APCs were co-cultured with T-cells for 4 days. In all figures, APCs were isolated from mice injected with either 50μg HEL or no HEL: all results of the latter are on baseline.