CD4+ FoxP3+ regulatory T cells confer infectious tolerance in a TGF-beta-dependent manner

Abstract

CD4(+)FoxP3(+) regulatory T (T reg) cells comprise a separate lineage of T cells that are essential for maintaining immunological tolerance to self. The molecular mechanism(s) by which T reg cells mediate their suppressive effects remains poorly understood. One molecule that has been extensively studied in T reg cell suppression is transforming growth factor (TGF)-beta, but its importance remains controversial. We found that TGF-beta complexed to latency-associated peptide (LAP) is expressed on the cell surface of activated but not resting T reg cells. T reg cell LAP-TGF-beta plays an important role in the suppression of the proliferation of activated T cells, but it is not required for the suppression of naive T cell activation. More importantly, T reg cell-derived TGF-beta could generate de novo CD4(+)FoxP3(+) T cells in vitro from naive precursors in a cell contact-dependent, antigen-presenting cell-independent and alpha(V) integrin-independent manner. The newly induced CD4(+)FoxP3(+) T cells are suppressive both in vitro and in vivo. Transfer of activated antigen-specific T reg cells with naive antigen-specific responder T cells to normal recipients, followed by immunization, also results in induction of FoxP3 expression in the responder cells. T reg cell-mediated generation of functional CD4(+)FoxP3(+) cells via this TGF-beta-dependent pathway may represent a major mechanism as to how T reg cells maintain tolerance and expand their suppressive abilities.

Figure 1.

LAP is expressed by activated T reg cells, and TGF-β is required to suppress CD4⁺CD25⁺FoxP3⁻ T cells. (A) Expression of LAP and FoxP3 on freshly isolated lymph node cells from C57BL/6 mice. (B) CD4⁺CD25⁻ (thick line histogram) and CD4⁺CD25⁺ T cells (thin line histogram) were activated for 3 d with plate-bound anti-CD3 and IL-2 and analyzed for LAP expression. Isotype control is depicted with a solid gray histogram. (C) Activated CD4⁺CD25⁺ T cells were incubated with (thick line histogram) or without (thin line histogram) 20 μg/ml LAP for 2 h at 4°C and analyzed for LAP expression. Isotype control is depicted with a solid gray histogram. (D) CD4⁺CD25⁺ T cells (5 × 10⁴/well) from lymph nodes of C57BL/6 mice were activated with plate-bound anti-CD3 or with irradiated T cell–depleted splenocytes (5 × 10⁴/well) and 0.25 μg/ml of soluble anti-CD3 in the presence or absence of LAP. [³H]TdR incorporation was determined after 72 h of culture. (E) CD4⁺CD25⁺ from C57BL/6 mice or CD4⁺CD25⁺ GFP⁺ T cells from C57BL/6 FoxP3-GFP knock-in mice were cultured together with irradiated T cell–depleted splenocytes (5 × 10⁴/well) and 0.25 μg/ml of soluble anti-CD3 in the presence or absence of LAP. [³H]TdR incorporation was determined after 72 h of culture. (F) CD4⁺CD25⁻ C57BL/6 T cells (5 × 10⁴/well) were co-cultured with CD4⁺CD25⁺ T cells from C57BL/6 mice (squares) or with CD4⁺CD25⁺GFP⁺ T cells from C57BL/6 FoxP3-GFP knock-in mice (circles) in the presence of irradiated T cell–depleted splenocytes (5 × 10⁴/well) and 0.25 μg/ml of soluble anti-CD3 with (open symbols) or without (closed symbols) 12.5 μg/ml LAP. [³H]TdR incorporation was determined after 72 h of culture. One representative experiment of at least three is shown (A–F).

Figure 2.

T reg cells confer infectious tolerance in a TGF-β–dependent manner. (A) Activated CD4⁺CD25⁻FoxP3⁻, CD4⁺CD25⁺FoxP3⁻, and CD4⁺CD25⁺FoxP3⁺ T cells were co-cultured with CFSE-labeled 5CC7 TCR transgenic RAG^−/− T cells, 2 μg/ml anti-CD3, splenic DCs, and IL-2 (100 U/ml) in the absence or presence of LAP. (B) Activated CD4⁺CD25⁺ and freshly isolated CD4⁺CD25⁺ were co-cultured with CFSE-labeled 5CC7 TCR transgenic RAG^−/− T cells, 2 μg/ml anti-CD3, splenic DCs, and IL-2 (100 U/ml) (left panels). Activated CD4⁺CD25⁺ T cells were co-cultured with the responders or separated from the responders in a transwell (right panel). (C) Activated CD4⁺CD25⁺ T cells from the thymus of 2-wk-old TGF-β1^−/− mice or from littermate controls were co-cultured with CFSE-labeled 5CC7 TCR transgenic RAG^−/− T cells. (D) Activated CD4⁺FoxP3⁺ T cells from peripheral lymph nodes of CD4cre-fur^f/f mice or littermate controls were co-cultured with CFSE-labeled 5CC7 TCR transgenic RAG^−/− T cells. Flow cytometric analysis was used to determine the level of FoxP3 expression after 4 d of the co-culture. All dot plots were gated on CFSE⁺ cells as shown in Fig. S2. One representative experiment out of at least two experiments is shown (A–D). Fig. S2 is available at http://www.jem.org/cgi/content/full/jem.20080308/DC1.

Figure 3.

The induction of FoxP3 expression by activated T reg cells is APC and α_v integrin independent. (A) CD4⁺CD25⁻ (left) and CD4⁺CD25⁺ (right) T cells from B10.A mice were activated for 4 d with plate-bound anti-CD3 and IL-2 (100 U/ml). The activated T cells were then co-cultured for an additional 4 d with an equal number of CFSE-labeled CD4⁺ T cells from 5CC7 TCR transgenic RAG^−/− mice in the presence of plate-bound anti-CD3 and IL-2. (B and C) CD4⁺CD25⁺ T cells were activated with plate-bound anti-CD3 and IL-2 for 4 d in the absence (left) or the presence (right) of blocking anti-α_v monoclonal antibodies (50 μg/ml), RGE peptide, or RGD peptide (10 μg/ml). The activated cells were then co-cultured with CFSE-labeled 5CC7 TCR transgenic T cells co-cultured in the presence or absence of the same inhibitors used in the first culture. FoxP3 expression was determined on day 4 of the co-culture. One representative experiment out of at least three is shown (A–C).

Figure 4.

Induced FoxP3⁺ T cells are suppressive in vitro and in vivo. (A) T reg cell–induced CD4⁺FoxP3⁺ T cells (open squares) were isolated as in Fig. S5 and tested for suppressive ability by co-culturing them with CD4⁺CD25⁻ T cells (5 × 10⁴/well) in the presence of irradiated T cell–depleted splenocytes (5 × 10⁴/well) and 0.25 μg/ml of soluble anti-CD3. Activated CD4⁺CD25⁻GFP⁻ T cells (closed squares) from C57BL/6 FoxP3-GFP mice were used as controls. Proliferation was measured by the incorporation of [³H]TdR after 72 h of culture. (B) CD4⁺GFP⁻ T cells (5 × 10⁵) from C57BL/6 FoxP3-GFP knock-in mice were transferred into C57BL/6 RAG^−/− mice with or without 1.5 × 10⁵ T reg cell–induced FoxP3⁺ T cells isolated as described in Fig. S5. The weight of all recipients was measured at 6 wk after cell transfer. P < 0.006 using Student's t test. (C) Hematoxylin and eosin–stained tissue sections from the colon of C57BL/6 RAG^−/− mice that received CD4⁺FoxP3⁻ T cells with (right) or without (left) the addition of T reg cell–induced CD4⁺FoxP3⁺ T cells. Bar, 100 μm. (D) CFSE-labeled CD4⁺CD25⁻CD44⁻ HA TCR transgenic T cells (0.5 × 10⁶) were transferred into B10.D2 mice with or without activated HA-specific TCR transgenic T reg cells (2.5 × 10⁶). 1 d later, all recipients were immunized with HA peptide in IFA, and draining lymph node cells were analyzed for FoxP3 expression 48 h after transfer. One representative experiment out of at least two is shown (A–D). Fig. S5 is available at http://www.jem.org/cgi/content/full/jem.20080308/DC1.