Increased frequency and suppression by regulatory T cells in patients with acute myelogenous leukemia

Abstract

Purpose: Regulatory CD4(+)CD25(high)Foxp3(+) T cells (Treg) control peripheral immune tolerance. Patients with cancer, including those with hematologic malignancies, have elevated numbers of Treg in the peripheral circulation and in tumor tissues. However, mechanisms of suppression and clinical significance of Treg, especially in patients with acute myelogenous leukemia (AML), has not been well defined.

Experimental design: We prospectively evaluated the phenotype, function, and mechanisms of suppression used by Treg in newly diagnosed untreated AML patients. The relationship between the frequency of circulating Treg and the disease status as well as treatment outcome was also evaluated.

Results: The percentage of circulating Treg was higher (P < 0.0001) and their phenotype was distinct in AML patients relative to normal controls. Suppression mediated by Treg coincubated with proliferating autologous responder cells was also higher (P < 0.001) in AML than that mediated by control Treg. Using Transwell inserts, we showed that interleukin-10 and transforming growth factor-beta1 production as well as cell-to-cell contact were necessary for Treg-mediated suppression. Also, the pretreatment Treg frequency predicted response to chemotherapy. Unexpectedly, patients who achieved complete remission still had elevated frequency of Treg, which mediated high levels of suppressor activity.

Conclusions: Treg accumulating in the peripheral circulation of AML patients mediate vigorous suppression via contact-dependent and contact-independent mechanisms. Patients with lower Treg frequency at diagnosis have a better response to induction chemotherapy. During the post-induction period, the Treg frequency and suppressive activity remain elevated in complete remission, suggesting that Treg are resistant to conventional chemotherapy.

Fig. 1

Increased frequency of CD4⁺CD25^high T cells in the peripheral blood of patients with AML before treatment. A, gating strategy used to identify the CD4⁺CD25^high T reg. CD4⁺ T cells with mean fluorescence intensity of CD25 expression ≥120 were classified as CD25^high as reported previously (26). Only CD4⁺CD25^high T cells with this or higher mean fluorescence intensity in PBMC have suppressor activity. B, percentages of CD4⁺CD25⁺ (left) and CD4⁺CD25^high (right) T cells in the peripheral blood of NC and AML patients. The percentage of Treg was increased in AML patients relative to that of NC P < 0.001. C, multicolor confocal microscopy shows the presence of CD4⁺CD25 ⁺Foxp3⁺ and CD4⁺CD25⁺NFAT1⁺ in the peripheral blood of a representative AML patient. Green, CD4⁺ cells ( and 4); red, CD25⁺ cells ( and 5); blue, Foxp3⁺ cells (2); blue, NFAT1⁺ cells (5). Merged pictures show CD4⁺CD25⁺ Foxp3⁺ (3) and CD4⁺CD25⁺NFAT1⁺ (6). Bar, 10 μm.

Fig. 2

Phenotypic characteristic of CD4⁺CD25^high Treg obtained from PBMC of AML patients and NC. A, gating strategy used to identify the CD4⁺CD25^high Treg and coexpression of Foxp3 in CD4⁺CD25^high cells. B, percentage of cells positive for various markers within the CD4⁺CD25^high T cell subsets in NC and AML patients. Asterisks, significant differences between NC and AML patients. *, P < 0.05.

Fig. 3

Suppression of proliferation by CD4⁺CD25^high Treg. A, CD4⁺CD25⁻ responder T cells (R) isolated from PBMC of a representative NC and an AML patient before any treatment. R cells were labeled with carboxyfluorescein diacetate succinimidylester and stimulated with OKT3, anti-CD28 antibody, and IL-2 in the absence of Treg (R alone). The addition of CD4⁺CD25^high Treg (S) from PBMC of NC or AML patient at different suppressor to responder cell ratios inhibited R cell proliferation, with the highest inhibition observed at the 1:1 suppressor to responder cell ratio. B, suppression of proliferation by CD4⁺CD25^high Treg in the newly diagnosed AML patients was significantly higher compared with NC. C, intracellular expression of TGF-β1 and IL-10 in NC and AML patients. Although the levels of expression were low, the expression was significantly different. The intracellular expression of TGF-β1 and IL-10 after stimulation with phorbol 12-myristate 13-acetate and ionomycin was significantly higher in the AML patients compared with NC. Asterisks, significant differences between NC and AML patients at P < 0.001.

Fig. 4

Suppression mediated by CD4⁺CD25^high T cells is cell contact and cytokine dependent. CD4⁺CD25^high T cells (S) from AML patients were coincubated with carboxyfluorescein diacetate succinimidylester-labeled autologous CD4⁺CD25⁻ T cells (R) responding to OKT3, anti-CD28 antibodies, and IL-2. Neutralizing anti-IL-10 and/or anti-TGF-β1 antibodies were added to the assays. A, in the absence of Transwell inserts, the addition of anti-IL-10 or anti-TGF-β1 antibody to cocultures resulted in a significant reduction of suppression; the addition of both neutralizing antibodies resulted in an even greater reduction of Treg-mediated suppression (P < 0.001). B, in the presence of Transwell inserts, suppression was reduced relative to cultures without inserts (as in A). Nevertheless, suppression levels remained at 40%, indicating that the cell-to-cell contact was in part necessary. When neutralizing antibodies to IL-10 or TGF-β1 were added to these cocultures, suppression was further reduced with the greatest inhibition of Treg suppression observed when Transwell inserts were combined with neutralizing anti-IL-10 and anti-TGF-β1 antibodies (P < 0.00001). Asterisks, significant differences between R + Treg and R + Treg + anti-IL-10, R + Treg + anti-TGF-β1, and R + Treg + anti-IL-10 + anti-TGF-β1 ± Transwell inserts.

Fig. 5

Constitutive expression of CD39 and CD73 by Treg and adenosine generation. A, expression levels of CD39 and CD73 proteins on CD4⁺CD25^high T cells in AML patients and NC. B, percentages of CD4⁺CD25^high T cells coexpressing CD39 and CD73 in the circulation of AML patients and NC. Mean ± SD (A and B). C, ATP hydrolysis by CD4⁺CD25⁺ or CD4⁺CD25⁻ cells isolated from a representative AML patient or a NC. Note that CD4⁺CD25⁺ T cells from AML hydrolyzed more exogenous ATP than CD4⁺CD25⁺ from NC or CD4⁺CD25⁻ T cells. In the presence of an inhibitor of ectonucleotidases, ARL67165, the ability of CD4⁺CD25⁺ cells to hydrolyze ATP was reduced. Representative of five experiments done. Asterisks, significant differences between triplicate samples tested at P < 0.001.

Fig. 6

Frequency and suppressor function of Treg in AML patients at diagnosis and after achieving CR. A, frequency of Treg was evaluated in all patients at diagnosis. Patients who achieved CR after induction chemotherapy had lower Treg levels compared with patients that had persistent leukemia. B, frequency of Treg after induction chemotherapy was evaluated in 7 patients who had achieved CR and was found to be significantly higher at the time of CR relative to that determined before treatment. C, compared with NC, the suppressor activity of CD4⁺CD25^high Treg remained elevated in AML patients who achieved CR. Elevated suppression levels were similar to those determined before treatment levels but were significantly higher relative to suppression mediated by Treg from NC. Mean ± SD. Asterisks, significant differences in suppression mediated by Treg of AML patients or NC at P < 0.001.