High donor FOXP3-positive regulatory T-cell (Treg) content is associated with a low risk of GVHD following HLA-matched allogeneic SCT

Abstract

Regulatory T cells (T(reg)s) that constitutively express FOXP3 are instrumental to the maintenance of tolerance and may suppress graft-versus-host disease (GVHD) in humans. To determine whether regulatory T cells in allogeneic stem cell transplants (SCTs) ameliorate GVHD after transplantation, we quantitated the coexpression of FOXP3 on CD4(+) T cells in 32 donor SCTs infused into HLA-matched siblings and examined GVHD incidence in respective recipients. High CD4(+)FOXP3(+) T-cell count in the donor was associated with a reduced risk of GVHD. We monitored T(reg)s during immune reconstitution in 21 patients with leukemia undergoing a T-cell-depleted allogeneic SCT. Early after SCT, there was a significant expansion in the CD4(+)FOXP3(+) T-cell compartment. A low CD4(+)FOXP3(+) T-cell count early after SCT (day 30) was associated with an increased risk of GVHD, and the ratio of CD4(+)FOXP3(+) T cells to CD4(+)CD25(+)FOXP3(-) T cells was significantly reduced in patients with GVHD, suggesting diminished control of effector T cells. Our findings suggest that graft T(reg) content may predict for risk of GVHD after SCT. Determining the T(reg) levels in the donor and manipulating T(reg)s early after transplantation may provide a new approach to controlling GVHD.

Figures

Figure 1.

Phenotypic characterization of regulatory T cells. Phenotypic analysis was performed on healthy donors (n = 32) and patients with leukemia (n = 21) followed longitudinally from baseline (before SCT), days +30, +45, +60, +90, and +120 after SCT. Representative data on one individual are presented here. (A) PBMCs were stained initially with anti-CD3, anti-CD4, and anti-CD25 antibodies followed by intracellular staining with anti–CTLA-4 and anti-FOXP3. (B) The histogram was gated on CD4+CD25+ T cells and represents the proportion of CD4+CD25+ T cells that are FOXP3+. (C) The dot plot was gated on CD4+ T cells: the upper right quadrant represents CD25+FOXP3+ T-cell population, and the lower right quadrant, the CD25–FOXP3+ population. (D-F) CTLA-4 and FOXP3: histograms are gated on CD4+FOXP3+ and CD4+FOXP3– populations. Whereas CD4+FOXP3+ T cells also co-express CTLA-4 (E), CD4+FOXP3– T cells were essentially CTLA-4 negative (F). (G) Correlation between FOXP3 mRNA copies and CD4+FOXP3+ (%). Dots correspond to individual samples tested from healthy donors and patients with leukemia. A significant positive correlation was seen between the frequency of CD4+FOXP3+ T cells and FOXP3 mRNA copies, as shown by the trendline.

Figure 2.

The Treg compartment is expanded in patients with leukemia prior to SCT compared to healthy donors. PBMCs were stained with monoclonal antibodies to CD3, CD4, and FOXP3, and the absolute and relative frequencies of CD4+ and CD4+FOXP3+ T cells were studied in patients with leukemia before SCT (□) and their respective donors (○) (A-B). PBMCs were obtained from donors prior to G-CSF administration for stem cell mobilization. In addition, we examined FOXP3 gene expression in purified CD4+ T cells from patients and donors (C). Patients with leukemia had fewer CD4+ T cells compared to healthy donors (n = 16) (A); however, the Treg compartment of patients with leukemia was relatively expanded as demonstrated by increased percentage of CD4+FOXP3+ T cells (n = 12) (B) and increased numbers of FOXP3 mRNA copies (n = 5) (C). (D) Patients were further stratified into high-risk (n = 11) (▪) and low-risk (n = 5) (□) disease as defined in Table 1. There was no significant association between FOXP3 mRNA expression and disease status. Bars represent median values. NS indicates not significant.

Figure 3.

Treg reconstitution after SCT. Using flow-based frequency enumeration and absolute lymphocyte counts acquired from a complete blood count obtained on the same day, relative and absolute numbers of CD4+CD3+, CD4+FOXP3–, and CD4+FOXP3+ were calculated for healthy donors (n = 21) (○), patients with leukemia at baseline, prior to allogeneic transplantation (n = 21) (pre; □), and patients after SCT (n = 21) at regular intervals: days +30 (▾), +45 (♦), +60 (•), +90 (▪), and +120 (▴). Bars indicate median values. Samples obtained following DLI are presented as symbols in red. Compared to healthy donors, on day 30 after SCT, circulating CD4+ and CD4+FOXP3– T-cell compartments were significantly reduced (A-B, D-E), whereas the percent of CD4+ subset composed of FOXP3+ cells was significantly increased (C). Bars represent median values.

Figure 4.

Effect of low-dose cyclosporine A (CSA) on D 30 CD4+ and CD4+FOXP3+ T-cell recovery. Patients who received low-dose CSA (▪) were compared to those who did not receive CSA (•) as immunoprophylaxis for the first 4 weeks after SCT. (A) Low-dose CSA had no significant effect on CD4+ T-cell and (B) CD4+FOXP3+ T-cell recovery. (C) Similarly, FOXP3 mRNA gene expression was not significantly different (NS) in the 2 groups. Bars represent median values.

Figure 5.

Inverse relationship between CD4+FOXP3+ and CD4+CD25+ T cells and acute GVHD. We compared CD4+, CD4+FOXP3+, and CD4+CD25+ T cells in donors of patients with no or grade 1 GVHD (n = 12) (○) and donors of patients with GVHD (n = 18) (•). We also examined CD4+, CD4+FOXP3+, and CD4+CD25+ T cells at days 30 to 45 after SCT in patients with grade 0 to 1 GVHD (n = 6) (□) and patients with grade 2 to 4 GVHD (n = 13) (▪). (A) The absolute CD4+ T-cell count was not significantly different in donors and patients with or without GVHD. (B) Donors of patients who did not develop GVHD had increased numbers of CD4+FOXP3+ T cells. Similarly, in patients with GVHD, after SCT, the absolute CD4+FOXP3+ T-cell count was significantly lower, whereas the absolute CD4+CD25+ T-cell count was significantly higher than those who did not develop GVHD (B-C). In addition, the proportion of CD4+CD25+ T cells that were FOXP3+ was significantly lower in patients with GVHD, indicating expansion of CD4+CD25+FOXP3– effector T cells (D). Bars indicate median values. Symbols for patient-donor pairs are color matched.