In vivo Treg suppression assays

Abstract

To fully examine the functionality of a regulatory T cell (T(reg)) population, one needs to assess their ability to suppress in a variety of in vivo models. We describe five in vivo models that examine the suppressive capacity of T(regs) upon different target cell types. The advantages and disadvantages of each model including resources, time, and technical expertise required to execute each model are also described.

Fig. 1

Gating profile for sorting T_regs and T_conv cells. The cells are first gated on live lymphocytes (not shown) and then a second gate is placed on the CD4⁺ cells (histogram). The CD4⁺ cells are further separated into either a CD45RB^high/CD25⁻(T_conv) gate or CD45RB^low/CD25⁺ (T_reg) gate.

Fig. 2

Procedures and analysis pertaining to the B16 melanoma and Foxp3⁻ rescue models. (a) The area on the flank of the mice that requires shaving and the location of the i.d. injection for the B16 melanoma model. (b) Sexing of a female (left ) and male (right ). (c) The proper technique recommended to hold a 2-day-old pup for i.p. injections. (d) The i.p. injection technique for a 2-day-old pup in the Foxp3⁻ rescue model. (e) Exterior of Foxp3⁻ (left (right ). mice that received no T_regs ) or wild-type natural T_regs (f) Spleens and lymph nodes of Foxp3⁻(left ) or wild-type mice that received no T_regs natural T_regs (right ). Black bar, 1 cm

Fig. 3

Microscopic H&E illustrations of Foxp3⁺ and Foxp3⁻ littermate mice of the lung, liver and ear pinna. (Lung ) The Foxp3⁺ mouse does not have inflammatory cells in or around either the bronchioles (B) or blood vessels (BV), and the interstitial septae (IS) are narrow, thin, and lack inflammatory cell infiltrates. In the Foxp3⁻ mouse, inflammatory cell infiltrates (*) surround bronchioles (B) and pulmonary blood vessels (PV) and focally thicken the interstitial septae (IS). (Liver ) The portal tracts (T) and liver lobular parenchyma (P) of the Foxp3⁺ mouse lack inflammatory infiltrates. Inflammatory cells fill some portal tracts (T) of the Foxp3⁻ mouse and they infiltrate the periportal hepatocytes broadening the portal tracts consistent with interface hepatitis. Foci of inflammatory cells (*) are randomly scattered through the liver lobular parenchyma of the Foxp3⁻ mouse. (Ear pinna) The dermis (D) and fatty (F) tissue of the Foxp3⁺ are void of inflammatory cells. Inflammatory cell infiltrates are present in a loose (*) and dense (**) pattern. The dermis is thicker and the fat tissue is obscured with inflammatory cell infiltrates in the Foxp3⁻ mouse.