Effective expansion of alloantigen-specific Foxp3+ CD25+ CD4+ regulatory T cells by dendritic cells during the mixed leukocyte reaction

Abstract

Thymic-derived CD25+ CD4+ T regulatory cells (Tregs) suppress immune responses, including transplantation. Here we evaluated the ability of dendritic cells (DCs) to expand alloantigen-specific Tregs in the mixed leukocyte reaction (MLR) that develops from polyclonal populations of T cells. The allogeneic DCs, when supplemented with IL-2 in the cultures, were much more effective than bulk spleen cells in expanding the numbers of Tregs. Likewise, DCs and not spleen cells were effective in sustaining expression of the transcription factor Foxp3 in Tregs, but neither IL-2 nor CD80/86 was required for this effect in the cultures. On a per-cell basis, the DC-expanded, but not unexpanded, Tregs were more potent suppressors of a fresh MLR by CD25- CD4+ T cells. Suppression was 3- to 10-fold more active for MLRs induced by the original alloantigens than for third-party stimulators. When DC-expanded Tregs were introduced into sublethally irradiated hosts, the T cells suppressed graft-versus-host-disease induced by CD25- CD4+ T cells. Again, suppression was more active against the same mouse strain that provided the DCs to expand the Tregs. Therefore, alloantigen-selected Tregs are more effective suppressors of responses to major transplantation antigens, and these Tregs can be expanded from a polyclonal repertoire by DCs.

Fig. 1.

Allogeneic DCs together with IL-2 expand Tregs. (A) BALB/c CD25⁺CD4⁺ T cells (10⁴) were cultured with 10⁴ B6 BM-DCs, selected as CD86 high mature DCs with or without LPS stimulation. Spleen CD11c⁺ DCs or 10⁵ splenic APCs (10⁴) were also tested. The DCs were added to T cells without (open) or with (closed) 100 units/ml rhIL-2. (B) As in A, 10⁴ BALB/c CD62L⁺ or CD62L⁻ CD25⁺ CD4⁺ T cells were cultured with 10⁴ B6 or BALB/c BM-DCs and IL-2 for 7 d. A and B are one of three similar results.

Fig. 2.

Foxp3 expression on expanded Tregs. (A) Expression of Foxp3 relative to isotype control antibody on CD4⁺ spleen cells labeled for CD25. (B) B6 Thy1.1⁺ CD25⁺ CD4⁺ T cells (10⁴) were cultured with 10⁴ BM-DCs or 10⁵ spleen cells from BALB/c mice with or without 500 units/ml IL-2 for 7 d. The cultures were gated on CD4⁺ and Thy1.1⁺ cells. (C) Viable cell numbers at 7 d and fold increases compared with cell numbers at day 0. (D) As in B, but Thy1.1⁺ BALB/c CD25⁺ CD4⁺ T cells were cultured with DCs from B6 control or CD80/CD86^−/− mice with or without 500 units/ml IL-2 for 7 d. One of three similar results.

Fig. 3.

Allogeneic-DC expanded Tregs suppress the MLR in an antigen-specific manner. CD62L⁺ CD25⁺ CD4⁺ T cells from Thy1.1⁺ B6 mice were freshly isolated or expanded with BALB/c or SJL-DCs. These were mixed with 10⁵ CFSE-labeled, Thy 1.2⁺, B6, and CD25⁻ CD4⁺ T cells in various ratios and stimulated with 10⁵ irradiated spleen cells from BALB/c (A) or SJL mice (B). Five days later, CFSE dilution was analyzed by FACS. The displayed cells were gated on live Thy1.2⁺ cells, and the percent of divided CFSE-low live Thy1.2⁺ cells is shown inside the plot. (C) As in B, but numbers of live Thy1.2⁺ responder cells per culture. One of two similar experiments.

Fig. 4.

Syngeneic DC-expanded Tregs are weak and antigen-nonspecific suppressors. (A) As in Fig. 3, CD25⁺ CD4⁺ T cells from CD45.2 B6 mice were expanded with allogeneic (BALB) or syngeneic (B6) DCs and added to 10⁵ CFSE-labeled, CD45.1⁺ CD25⁻ CD4⁺ B6 responder T cells in various ratios. These were stimulated with 3 × 10⁵ irradiated BALB/c (Left) or CBA (Right) spleen cells. The displayed cells were gated on live responder CD45.1⁺ cells, and the percent live CD45.1⁺ divided cells is shown inside the plot. The numbers of live CD45.1⁺ cells per culture are also shown for BALB/c (B) and CBA (C) MLR data. One of five similar experiments.

Fig. 5.

Allogeneic DC-expanded Tregs suppress GVHD in an antigen-specific manner. (A) Naïve B6 CD25⁻ CD4⁺ T cells (10⁵) (effectors alone, closed circles) were adoptively transferred into irradiated bm12 recipients with or without 10⁵ CD62L⁺ CD25⁺ CD4⁺ T cells that had been expanded with bm12-DC (alloantigen-specific, open triangles) or CBA-DC (third-party, open squares). Percent of surviving mice is shown (n = 5). (B) Naïve B6 CD25⁻ CD4⁺ T cells (2 × 10⁵) (effector-alone, closed circles) were adoptively transferred into irradiated BALB/c recipients with or without 2 × 10⁵ CD62L⁺ CD25⁺ CD4⁺ T cells that had been expanded with BALB DC (alloantigen-specific, open triangles) or B6-DC (syngeneic, antigen-nonspecific, open diamonds) (n = 5). (C) As in B, but CD62L⁺ CD25⁺ CD4⁺ T cells that had been expanded with BALB DC-expanded (alloantigen-specific, open triangles), or CBA DC-expanded (third party, open squares) were cotransferred (n = 6). A and B are each representative of two experiments, whereas C is a single experiment.