Recruitment of Foxp3+ T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor

Abstract

Although certain chemokines and their receptors guide homeostatic recirculation of T cells and others promote recruitment of activated T cells to inflammatory sites, little is known of the mechanisms underlying a third function, migration of Foxp3(+) regulatory T (T reg) cells to sites where they maintain unresponsiveness. We studied how T reg cells are recruited to cardiac allografts in recipients tolerized with CD154 monoclonal antibody (mAb) plus donor-specific transfusion (DST). Real-time polymerase chain reaction showed that intragraft Foxp3 levels in tolerized recipients were approximately 100-fold higher than rejecting allografts or allografts associated with other therapies inducing prolonged survival but not tolerance. Foxp3(+) cells were essential for tolerance because pretransplant thymectomy or peritransplant depletion of CD25(+) cells prevented long-term survival, as did CD25 mAb therapy in well-functioning allografts after CD154/DST therapy. Analysis of multiple chemokine pathways showed that tolerance was accompanied by intragraft up-regulation of CCR4 and one of its ligands, macrophage-derived chemokine (CCL22), and that tolerance induction could not be achieved in CCR4(-/-) recipients. We conclude that Foxp3 expression is specifically up-regulated within allografts of mice displaying donor-specific tolerance, that recruitment of Foxp3-expressing T reg cells to an allograft tissue is dependent on the chemokine receptor, CCR4, and that, in the absence of such recruitment, tolerizing strategies such as CD154 mAb therapy are ineffectual.

Figure 1.

Post-Tx Foxp3 expression. (a) qPCR analysis showed a progressive decrease in splenic Foxp3 mRNA (left, mean ± SD) and an increase in associated cardiac grafts (right); expression shown relative to levels at 7 or 3 d post-Tx, respectively. (b) Comparison at 7 d post-Tx showed increases in allograft Foxp3 mRNA (qPCR, mean ± SD) were >5-fold that of C57BL/6 isografts (left), whereas levels in grafts from recipients treated with CD154 mAb/DST therapy were >100-fold higher than control grafts (right); expression in each panel is relative to native heart. (c) Confirmation of intragraft Foxp3 mRNA expression by Northern analysis, comparing expression in control (C) normal hearts, acutely rejecting (day 7) allografts or serially harvested isografts versus allografts from hosts treated with CD154 mAb/DST as indicated. (d) Foxp3 staining was exceedingly rare in allografts undergoing acute rejection (left), but readily detected as Foxp3⁺ mononuclear cells in mice treated with CD154 mAb/DST; immunoperoxidase labeling of hematoxylin-stained cryostat sections. Original magnification, ×250 (inset shows lack of staining with Foxp3 peptide-absorbed antibody).

Figure 2.

Foxp3, T reg cell, and costimulation blockade. (a) CD154/DST induced long-term cardiac allograft survival and donor-specific tolerance, whereas CD28^−/− recipients treated with neutralizing anti-ICOS mAb also accepted primary cardiac allografts long term but rejected second donor allografts. Inset shows how thymectomy and CD25 mAb therapy prevents long-term allograft survival despite CD154/DST therapy (left), and that CD25⁺ cells are also required for maintenance of allograft survival (right) because CD25 mAb therapy beginning at day 35 restores allograft rejection. (b) Levels of Foxp3 and CD3 mRNA (qPCR, mean ± SD) in day 7 cardiac allografts. (c) Flow cytometry showing decreased CD4⁺CD25⁺ cells in thymic and splenic samples of control, CD28^−/− or ICOS^−/− mice; numbers are percentage of CD4⁺CD25⁺ cells within gates set using an CD25 isotype mAb. (d) Analysis of corresponding Foxp3 mRNA (qPCR) with expression shown relative to levels in samples from CD28^−/− mice; results are representative of three independent experiments.

Figure 3.

Intragraft mRNA expression of immunoregulatory molecules and chemokine pathways at day 7 post-Tx. (a) Intragraft levels of CD25, GITR, TGF-β, and IL-10 did not correlate with selective high Foxp3 expression in CD154 mAb/DST grafts. (b) Comparison of intragraft chemokine receptor expression. (c) Intragraft expression of CCR4 (MDC, TARC) and CCR8 (TCA3) ligands. qPCR results, which are shown relative to expression in native hearts, are representative of three independent experiments.

Figure 4.

Key role of CCR4 in T reg cell recruitment and tolerance induction. (a) qPCR analysis of Foxp3, CCR4, and CCR8 mRNA expression by CD4⁺CD25⁺ versus CD4⁺CD25⁻ T cells; cells were isolated using magnetic beads to >95% purity. Data are expressed as relative to levels in CD4⁺CD25⁻ cells and are representative of three separate experiments. (b) Targeting of TCA3 using a neutralizing mAb had no effect on allograft rejection in wild-type recipients or long-term allograft survival when administered in conjunction with CD154 mAb/DST therapy. In contrast, CCR^−/− recipients rejected allograft normally and could not be tolerized using CD154 mAb/DST therapy. (c) Analysis by qPCR of intragraft levels of Foxp3, CD3, and CD25 mRNA expression at day 7 post-Tx, showing marked reduction in intragraft Foxp3 but not CD3 or CD25 in CCR4^−/− versus control mice treated with CD154 mAb/DST; results are shown relative to expression in native hearts and are representative of three independent experiments.

Figure 5.

Numbers and function of T reg cells in CCR4^−/− mice are comparable to WT controls. (a) FACS analysis of CD25⁺ expression by CD4⁺ cells from naive thymus, and corresponding qPCR analysis of Foxp3 mRNA expression in whole thymus from WT and CCR4^−/− mice. (b) FACS analysis of CD25⁺ expression by CD4⁺ cells in naive spleen and corresponding qPCR analysis of Foxp3 mRNA expression in whole spleen. (c) CFSE- labeled CD4⁺CD25⁻ responder T cells exhibited decreased CD3 mAb- induced CFSE proliferation in the presence of CD4⁺CD25⁺ T reg cells from either WT or CCR4^−/− mice at the indicated ratios, as compared with no T reg cells added. (right) Histograms show the results of quantitation of the absolute number of responder CD4⁺CFSE⁺ cells in the absence and presence of T reg cells from WT and CCR4^−/− mice.