Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents

Abstract

Immune thrombocytopenia (ITP) is an autoantibody-mediated bleeding disorder with both accelerated platelet destruction and impaired platelet production. We and others have described impaired regulatory CD4(+)CD25(hi) T cells (Treg) numbers and/or suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients, but information on the immunologic responses of treated patients are lacking. We studied the immunologic profile of chronic ITP patients before (n = 10) and during treatment with thrombopoietin receptor (TPO-R) agonists (n = 9). Treg activity, as measured by suppression of proliferation of autologous CD4(+) CD25(-) cells, was improved in patients on treatment (P < .05), and the improvement correlated with reduction in interleukin-2-producing CD4(+) cells, consistent with dampening of immune responses. There was a concomitant increase in total circulating transforming growth factor-β1 (TGF-β1) levels (P = .002) in patients on treatment, and the levels of TGF-β1 correlated with the degree of improvement in platelet counts (r = .8, P = .0002). This suggests that platelets in patients on TPO-R treatment may play a role in improving Treg function, either directly or indirectly by enhanced release of TGF-β1 as a result of greater platelet turnover. In conclusion, our findings suggest that thrombopoietic agents in patients with ITP have profound effects to restore immune tolerance.

Figure 1

Platelet counts in patients before and on treatment with thrombopoietic agents. Platelet counts are shown for the same day that blood was sampled for immunologic analysis. Platelet counts of patients on treatment (■) were statistically higher (P = .002) compared with before treatment (●). Median pretreatment count was 22 × 10⁹/L, interquartile range 11 to 30 × 10⁹/L versus 105 × 10⁹/L, interquartile range 44 to 243 × 10⁹/L on treatment group.

Figure 2

Frequency of CD25^hiFoxp3⁺ in the patient cohort. (A) Whole blood was stained with anti-CD4 peridinin chlorophyll protein and anti-CD25 allophycocyanin, followed by intracellular staining using phycoerythrin-conjugated anti-Foxp3. The cells were first gated on the CD4⁺ lymphoid population in peripheral blood mononuclear cells, and the frequency of CD25^hiFoxp3⁺ cells was measured based on the shown gating strategy. (B) Percentage of CD25^hiFoxp3⁺ in the CD4⁺ population in patients before (●) and on treatment with thrombopoietic agents (■) as well as the normal healthy controls (♦). No statistical differences among the groups were seen.

Figure 3

Circulating Treg-suppressive activity in patients before and on treatment with thrombopoietic agents. Suppression of proliferation by CD4⁺CD25^hi Treg was analyzed in patients before treatment (n = 10) and in patients on treatment with thrombopoietic agents (n = 9) as well as normal healthy controls (n = 9). Sorted populations of CD4⁺CD25⁻ T cells were stimulated with plate-bound 0.1 μg/mL anti-CD3 antibodies and allogeneic accessory cells, alone or cocultured at various ratios (solid lines indicate 1:1; and dotted lines, 1:4) with autologous sorted CD4⁺CD25^hi cells, and mean inhibition was calculated as described in “Proliferation/Treg cell suppression assay.” Although this is a cross-sectional study with different patients in the pretreatment and on treatment groups, we have joined the pretreatment and on treatment data points by a line (solid or dotted) to highlight the difference in the suppressive activity between the 2 groups. Suppression measured at a 1:1 ratio of CD4⁺CD25⁻ to CD4⁺CD25^hi in patients before treatment was 53% ± 7% but was higher in patients on treatment (63% ± 4%). Similarly, at 1:4, the pretreatment group had a lower suppressive activity (35% ± 7%) compared with on treatment group of 51% ± 6% (overall P = .045). For comparison, the healthy volunteer controls' suppressive activity at 1:1 ratio (70% ± 2%) and at 1:4 ratio (50% ± 5%) is also indicated. The raw proliferation data are shown in supplemental Figure 2.

Figure 4

Correlative analysis of Treg functional activity and frequency of IL-2–expressing sorted CD4⁺ T cells. Sorted CD4⁺CD25⁻ T cells from the patient cohort were stimulated with phorbol myristate acetate and ionomycin for 5 hours in the presence of protein transport inhibitor, and intracellular expression of IL-2 was measured by flow cytometry. The frequency of IL-2-expressing CD4⁺ cells negatively correlated with the patients' Treg-suppressive activity at 1:1 ratio of CD4⁺CD25⁻ and CD4⁺CD25^hi (r = −0.47, P = .04).

Figure 5

Correlative study of platelet counts and TGF-β1 levels. Circulating total TGF-β1 levels of the patient cohort from Table 1 were measured by ELISA using platelet-poor plasma. Correlative analysis indicated a strong positive association between the patients' platelet counts and TGF-β1 levels (r = 0.8, P = .0002).

Figure 6

Circulating TGF-β1 levels in patients before and on treatment with thrombopoietic agents. Plasma levels of TGF-β1 were significantly increased in patients on treatment (■) compared with before treatment (●, P = .002, using the rank order Mann-Whitney test). As comparison, levels of TGF-β1 in normal healthy controls are shown (♦).

Figure 7

sCD40L levels in platelet-poor plasma of patient cohort. Circulating levels of sCD40L in platelet-poor plasma were significantly lower in patients on treatment (■) compared with pretreatment (●, P = .02). As comparison, levels of sCD40L in normal healthy controls are shown (♦).