Selective elimination of human regulatory T lymphocytes in vitro with the recombinant immunotoxin LMB-2

Abstract

CD4(+)CD25(+) T-regulatory cells (T(reg)) can inhibit the proliferation and cytokine secretion of CD4(+)CD25(-) helper T cells in mice and humans. In murine tumor models, the presence of these T(reg) cells can inhibit the antitumor effectiveness of T-cell transfer and active immunization approaches. We have thus initiated efforts to eliminate T(reg) cells selectively from human peripheral blood mononuclear cells (PBMCs) to potentially bolster antitumor responses. LMB-2 is a recombinant immunotoxin that is a fusion of a single-chain Fv fragment of the anti-Tac anti-CD25 monoclonal antibody to a truncated form of the bacterial Pseudomonas exotoxin A. In vitro incubation of human PBMCs with LMB-2 reduced the levels of CD4(+)CD25(+) and Foxp3-expressing cells without impairing the function of the remaining lymphocytes. The short in vivo half-life of LMB-2 makes it an attractive candidate for reducing human T(reg) cells in vivo before the administration of cancer vaccine or cell transfer immunotherapy approaches.

FIGURE 1

Varying doses of LMB-2 were incubated with resting human PBMCs for 48 hours, and the percentages of residual CD4⁺CD25⁺ cells (top) and the copies of Foxp3 mRNA per 10⁴ copies of β-actin mRNA (bottom) were evaluated. A dose-related reduction in these 2 surrogate markers of human T_reg cells was seen.

FIGURE 2

Resting human PBMCs were incubated in vitro for 48 hours with 100 ng/mL of LMB-2, LMB-9 (an immunotoxin that recognized the Lewis Y antigen not expressed on PBMCs), LMB-2^Asp553 (anti-CD25 fused to an inactivating mutation in the Pseudomonas exotoxin), or the intact anti-Tac mAb. Some decrease in the percentage of CD4⁺ CD25⁺ cells and/or Foxp3-expressing cells was seen with LMB-9, LMB-2^Asp553, and anti-Tac. A greater decrease was seen in PBMCs treated with LMB-2, although it did not reach the level of Foxp3 depletion achieved by ex vivo mechanical depletion using anti-CD25 magnetic leads.

FIGURE 3

Resting human PBMCs were incubated with LMB-2 for 48 hours, and the percentage of CD4⁺CD25⁺ cells remaining was evaluated by fluorescence-activated cell sorter analysis. A significant decrease in CD25-expressing cells was seen as a result of LMB-2 treatment in whole PBMCs and negatively isolated purified CD4⁺CD25⁺ cells (top). A similar decrease was seen in a second experiment testing negatively and positively isolated CD4⁺ cells (bottom).

FIGURE 4

Same cells as in Figure 3. LMB-2 treatment also reduced Foxp3 levels in PBMCs and in purified CD4⁺ cells.