Repeated Long-Term DT Application in the DEREG Mouse Induces a Neutralizing Anti-DT Antibody Response

Abstract

Regulatory T (Tregs) cells play an important role in mediating tolerance to self-antigens but can also mediate detrimental tolerance to tumours and pathogens in a Foxp3-dependent manner. Genetic tools exploiting the foxp3 locus including bacterial artificial chromosome- (BAC-) transgenic DEpletion of REGulatory T cells (DEREG) mice have provided essential information on Treg biology and the potential therapeutic modulation of tolerance. In DEREG mice, Foxp3⁺ Tregs selectively express enhanced green fluorescent protein (eGFP) and diphtheria toxin (DT) receptor, allowing for the specific depletion of Tregs through DT administration. We here provide a detailed overview about an important consideration that long-term administration of DT induces a humoral immune response with an appropriate production of anti-DT antibodies that can inactivate DT and thus abrogate its effect in the DEREG mouse. Additionally, we showed that anti-DT mouse serum partially neutralized DT-induced Foxp3 inhibition.

Figure 1

Experimental design, Foxp3, and anti-DT antibody follow-up during chronic DT treatment. (a) Schematic presentation of the experimental design. Foxp3⁺ Treg cell depletion was achieved by intraperitoneal (i.p.) administration of 110 ng diphtheria toxin (DT) 3 times per week for 1, 2, 3, and 4 weeks. DT administration was stopped after 4 weeks; Foxp3 and anti-DT antibody were determined at indicated time-points. (b) Titrated DT was injected i.p. at 0, 75, 150, and 300 ng; Foxp3 was determined by flow cytometry at day 1 and day 3 after DT injection. (c) 110 ng DT was injected i.p. 3 times per week at 1, 2, 3, and 4 weeks and then stopped. Foxp3 was determined by flow cytometry at week 4 and week 16. (d) Serum levels of anti-DT antibody were determined by using ELISA at indicated time-points. The mice were aged 8 weeks when used to start the initial DT treatment. ^∗∗P < 0.01.

Figure 2

Anti-DT serum blocking. (a) Representative images of Foxp3⁺ T cells within CD4⁺ T cells with/without anti-DT serum blocking. (b) Frequency of Foxp3⁺ T cells within CD4⁺ T cells with/without anti-DT serum blocking. Six wild type C57BL6 mice were injected 110 ng DT i.p. 3 times per week and maintained for another 4 weeks. Mice were sacrificed and blood was taken from heart puncture. Serum anti-DT antibody levels were detected by ELISA. Seropositive samples were pooled, and from this pool 200 μL anti-DT serum was injected i.p. per mouse, and subsequently 110 ng DT/mouse was injected i.p. one day later. All the animals were sacrificed at the 3rd day, and CD4⁺Foxp3⁺ frequency in the spleen was measured by flow cytometry. ^∗P < 0.05.