Differential effects of IL-2 and IL-21 on expansion of the CD4+ CD25+ Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia

Abstract

CD4(+) CD25(+) regulatory T cells are expanded in solid and hematological malignancies including chronic lymphocytic leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin (IL)-21 on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory T cell (Treg) induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4(+)CD25(High) regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory T cells or induced expression of Foxp3 in CD4(+)CD25(Intermediate) or CD4(+)CD25(Dim/-) T cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4(+)CD25(+)Foxp3(+)T cells, IL-2 and IL-21 exhibited a redundant role in Treg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.

Figure 1

Analysis of expression of IL-21 receptor in CD4⁺CD25⁺ Regulatory Tt cells from CLL patients. PpBMC isolated from CLL patient samples were stained with anti-CD4 (APpC), anti-CD25 (FITtC) and anti-IL-21Receptor (PEpe) antibodies and analyzed by FACS. Tthe left panel shows CD4 and CD25 staining profiles with the boxed regions indicating CD25^high, CD25^Intermediate and CD25^dim/− cells that are further analyzed for IL-21 receptor expression. histogram on the right shows the IL-21 receptor expression in CD4⁺CD25^High, CD4⁺CD25^Intermediate and CD4⁺ CD25^Dim/− populations shown on the left panel. results are representative of six independent experiments.

Figure 2

IL-2 but not IL-21 induces expansion of regulatory T cells. Whole blood samples from four independent CLL patients were treated with IL-2 or IL-21 (10 ng/ml) for 48 Hours. The cells were analyzed by tricolor staining using anti-CD4 (APC), anti-CD25 (FITC) and anti-Foxp3 (PE) antibodies. changes in (A) CD4⁺CD25^HighFoxp3⁺ cells (B) Right Panel shows CD4 and CD25 staining profiles with the boxed regions indicating CD25^High, CD25^Intermediate, CD25^dim/− Cells that are further analyzed for Foxp3 expression. histogram on the left panel shows the Foxp3 expression in CD4⁺CD25^High, cells in untreated, IL-2 (10 ng/ml) or IL-21 (10 ng/ml) treated PpBMC. (C and D) CD⁴+CD25^intermediateFoxp3⁺ (Ee and F) CD4⁺CD25d^im/-Foxp3⁺ normalized to media treated controls is shown. IL-2 and IL-21 effects when compared independently to untreated control, the expansion of CD4⁺CD25^High cells was significant only with IL-2 (Using Holms procedure estimated difference 1.39, CI 0.63, 2.14, p = 0.0057) in contrast to IL-21 (estimated difference 0.75, CI -0.01, 1.15 and p = 0.1048).

Figure 3

Treg induced suppression of NK mediated ADCC. Chromium-51 labeled Raji cells were incubated with rituximab, isotype control trastuzumab or no antibody. NK-92 cells were co-cultured with cells for 24 hours and then incubated with antibody coated Raji cells at 37°C for 4 hours. Percent relative cytotoxicity was measured by calculating Cr-51 release, normalized by minimum and maximum release. (A) shows the effect of Tregs on NK cell mediated ADDC. (B-D) show abrogation of NK mediated ADCC by that were pretreated for 72 hrs with immobilized anti-CD3 (B), IL-2 (C) or IL-21 (D). The results are the representative of three independent experiments.