Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells

Abstract

Chemokines dictate regional trafficking of functionally distinct T cell subsets. In rodents and humans, a unique subset of CD4(+)CD25(+) cytotoxic T lymphocyte antigen (CTLA)-4(+) regulatory T cells (Treg) has been proposed to control peripheral tolerance. However, the molecular basis of immune suppression and the trafficking properties of Treg cells are still unknown. Here, we determined the chemotactic response profile and chemokine receptor expression of human blood-borne CD4(+)CD25(+) Treg cells. These Treg cells were found to vigorously respond to several inflammatory and lymphoid chemokines. Treg cells specifically express the chemokine receptors CCR4 and CCR8 and represent a major subset of circulating CD4(+) T cells responding to the chemokines macrophage-derived chemokine (MDC)/CCL22, thymus and activation-regulated chemokine (TARC)/CCL17, I-309/CCL1, and to the virokine vMIP-I (ligands of CCR4 and CCR8). Blood-borne CD4(+) T cells that migrate in response to CCL1 and CCL22 exhibit a reduced alloproliferative response, dependent on the increased frequency of Treg cells in the migrated population. Importantly, mature dendritic cells preferentially attract Treg cells among circulating CD4(+) T cells, by secretion of CCR4 ligands CCL17 and CCL22. Overall, these results suggest that CCR4 and/or CCR8 may guide Treg cells to sites of antigen presentation in secondary lymphoid tissues and inflamed areas to attenuate T cell activation.

Figure 1

Chemotactic response profile of human CD4⁺CD25⁺ Treg cells. (a) Specific migration of human CD4⁺CD25⁺ T cells in response to CCL1, CCL22, CCL17, vMIP-I, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL11/Eotaxin, CCL19/ELC, CCL20/MIP-3α, CXCL11/I-TAC, CXCL12/stromal cell–derived factor (SDF)-1, and CXCL13/B lymphocyte chemoattractant (BLC) (1 μg/ml). Mean and SD n = 5 experiments for CCL1, CCL17, and CCL22, n = 3 for other chemokines, *P < 0.01. (b) Chemotactic responses of CD25⁻, CD25⁻CD45RO⁺ and CD25⁺CD45RO⁺CD4⁺ T cells to CCL1, CCL22, CCL17, CXCL11, CCL19, and CCL20. Filled circles (•) represent CD4⁺CD25⁺CD45RO⁺ T cells, whereas open circles (○) represent CD4⁺CD25⁻CD45RO⁺ T cells and open squares (□) represent CD4⁺CD25⁻ T cells. Mean and SD of one representative experiment of three performed in triplicate.

Figure 1

Chemotactic response profile of human CD4⁺CD25⁺ Treg cells. (a) Specific migration of human CD4⁺CD25⁺ T cells in response to CCL1, CCL22, CCL17, vMIP-I, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL11/Eotaxin, CCL19/ELC, CCL20/MIP-3α, CXCL11/I-TAC, CXCL12/stromal cell–derived factor (SDF)-1, and CXCL13/B lymphocyte chemoattractant (BLC) (1 μg/ml). Mean and SD n = 5 experiments for CCL1, CCL17, and CCL22, n = 3 for other chemokines, *P < 0.01. (b) Chemotactic responses of CD25⁻, CD25⁻CD45RO⁺ and CD25⁺CD45RO⁺CD4⁺ T cells to CCL1, CCL22, CCL17, CXCL11, CCL19, and CCL20. Filled circles (•) represent CD4⁺CD25⁺CD45RO⁺ T cells, whereas open circles (○) represent CD4⁺CD25⁻CD45RO⁺ T cells and open squares (□) represent CD4⁺CD25⁻ T cells. Mean and SD of one representative experiment of three performed in triplicate.

Figure 2

Immunoregulatory activity of CD25⁺ T cells migrated in response to CCL1 or CCL22. (a) Enrichment of immunosuppressive activity amongst CD4⁺ T cells migrated in response to CCL1 or CCL22. Alloantigen specific proliferative responses of CD4⁺ T cells that migrated in response to CCL1, CCL22, and CCL19 were analyzed in triplicate as described in Materials and Methods. *P = 0.05. (b) Reduction of immunosuppressive activity by depletion of CD25⁺ T cells from CD4⁺ T cells migrated in response to CCL1 or CCL22. After migration of CD4⁺ T cells in response to CCL1 or CCL22, the cells were split and one part was depleted of CD25⁺ T cells by immunomagnetic bead sorting. CD25⁺-depleted (CD25 depleted) and nondepleted populations were then analyzed for their alloantigen specific proliferative response.

Figure 2

Immunoregulatory activity of CD25⁺ T cells migrated in response to CCL1 or CCL22. (a) Enrichment of immunosuppressive activity amongst CD4⁺ T cells migrated in response to CCL1 or CCL22. Alloantigen specific proliferative responses of CD4⁺ T cells that migrated in response to CCL1, CCL22, and CCL19 were analyzed in triplicate as described in Materials and Methods. *P = 0.05. (b) Reduction of immunosuppressive activity by depletion of CD25⁺ T cells from CD4⁺ T cells migrated in response to CCL1 or CCL22. After migration of CD4⁺ T cells in response to CCL1 or CCL22, the cells were split and one part was depleted of CD25⁺ T cells by immunomagnetic bead sorting. CD25⁺-depleted (CD25 depleted) and nondepleted populations were then analyzed for their alloantigen specific proliferative response.

Figure 3

Expression of chemokine receptors CCR4 and CCR8 on CD25⁺ Treg cells. (a) Cell surface expression of CCR4 and CCR8 on CD25⁺ Treg cells. Purified CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were fixed, incubated with anti-CD4, anti-CCR4, or anti-CCR8 Abs, stained with the appropriate fluorochrome-conjugated secondary Abs, and analyzed by confocal microscopy. NC, not stained control. (b) Graphs illustrate the percentage of CCR4⁺ or CCR8⁺CD4⁺ T cells counted in each subpopulation in five different fields. P values are indicated. (c) Synergistic chemotactic response to CCL1 and CCL22 of Treg cells. CCL1 (100 ng/ml) and CCL22 (10 ng/ml) were used separately or in combination. Results are shown as chemotactic index for Treg cells. (d) CCR8 mediates migration of Treg cells in response to CCL1. The percentage of inhibition of specific migration of Treg cells in response to CCL1 and CCL22 (0.5 μg/ml) in the presence of MC148 (2 μg/ml) is shown.

Figure 4

Production of CCL17 and CCL22 by mature human DCs leading to preferential attraction of CD25⁺ Treg cells. Human monocyte-derived DCs were washed and stimulated with LPS. DCs were washed again after 16 h and cultured for additional 24 h before harvesting the supernatants. Supernatants were tested for their chemotactic activity on CD4⁺ T cells. Chemotactic index of CD4⁺CD25⁺ (black bars) and CD4⁺CD25⁻ (white bars) T cells is indicated. Where indicated, isotype-matched control (ctrl) or anti-CCL17 and/or anti-CCL22 mAbs (2 μg/ml) were added to the supernatants to remove the investigated chemokine as described in Materials and Methods. Migration to CCL22 is shown for comparison. Results are from one representative experiment of two performed.