Adjuvant effect of diphtheria toxin after mucosal administration in both wild type and diphtheria toxin receptor engineered mouse strains

Abstract

The finding that murine and simian cells have differential susceptibility to diphtheria toxin (DTx) led to the development of genetically engineered mouse strains that express the simian or human diphtheria toxin receptor (DTR) under the control of various mouse gene promoters. Injection of DTx into DTR engineered mice allows for rapid and transient depletion of various cell populations. There are several advantages to this approach over global knockout mice, including normal mouse development and temporal control over when cell depletion occurs. As a result, many DTR engineered mouse strains have been developed, resulting in significant insights into the cell biology of various disease states. We used Foxp3(DTR) mice to attempt local depletion of Foxp3+ cells in the lung in a model of tolerance breakdown. Intratracheal administration of DTx resulted in robust depletion of lung Foxp3+ cells. However, DTx administration was accompanied by significant local inflammation, even in control C57Bl/6 mice. These data suggest that DTx administration to non-transgenic mice is not always an immunologically inert event, and proper controls must be used to assess various DTx-mediated depletion regimens.

Keywords: Diphtheria toxin; Lung; Regulatory T cells; Tolerance.

Figure 1

Depletion of T regulatory cells is accompanied by increased inflammation after intratracheal DTx administration in Foxp3^DTR mice. Foxp3^DTR and C57Bl/6 mice were given three consecutive daily doses of 100ug Grade V OVA to induce inhaled tolerance. Ten days after the last dose of OVA, control PBS or DTx were given i.t. to tolerized mice. A) Representative flow cytometry gating strategy for identification of Tregs. After scatter gating, CD3+CD4+ T cells were identified. The gating of Foxp3+ cells is shown in the bottom left panel; CD25 expression on gated CD3+CD4+Foxp3+ cells is shown in the bottom right panel. B) BAL cell recovery in tolerized C57Bl/6 mice that received control PBS or DTx. Data are 48hr post treatment. C–D) Organ cell recovery (circles) and number of CD3+CD4+Foxp3+ cells (squares) in Foxp3^DTR mice are reported for BAL (A) and MLN (B) each of the first four days after DTx administration. For organ cell count data, n=3–4 per group per day. For Foxp3+ cell quantification, all are n=3–4 per group per day, but Control group was pooled. Percentages in A) are percent reduction from Control group. * = p<0.05 compared to Control.

Figure 2

Intratracheal DTx administration induces pulmonary inflammation in both Foxp3^DTR and control C57Bl/6 mice after OVA sensitization and challenge. A) Mice were tolerized and then received DTx or control treatments as in Figure 1. Following a single DTx treatment, mice were given 100ug OVA and 100ng LPS by o.p. route three consecutive days, followed two weeks later by twice daily OVA aerosol challenges for three consecutive days. Mice were sacrificed two days after the last challenge. B) Foxp3^DTR and C57Bl/6 mice were sensitized and challenged as in Figure 2A; DTx from Calbiochem was used to deplete Foxp3+ cells. BAL cell recovery was determined by Trypan blue exclusion on day 30. C) Same experimental setup as Figure 2B, but DTx from Sigma was used and additional control groups were included (see legend at the bottom of the Figure). Data are n=3–5 for each group. * = p<0.05.