Systemic IL-2 is increased in the absence of Treg. (A) CD45.1⁺ OT-I T cells were transferred to C57BL/6 mice immunized with OVA/QuilA and at the indicated time points, expression of CD25 and CD122 by OT-I T cells was determined by FACS. (B and D) CD45.1⁺ OT-I T cells were transferred to C57BL/6 mice treated with anti-CD25 or isotype control mAb and immunized or not with OVA/QuilA at the time of transfer. Seven days later, spleens were collected and the number of IL-2–producing OT-I T cells determined by intracellular cytokine staining and a FACS counting assay (B) or serum collected and IL-2 concentration determined by ELISA (D). (C and E) CD45.1⁺ OT-I T cells were transferred to anti-CD25 or isotype-treated nTx or sham-Tx 11c.OVA mice. Three days later, spleens were collected and the number of IL-2–producing OT-I T cells determined by intracellular cytokine staining and a FACS counting assay (C) and serum collected, and IL-2 measured by ELISA (E). Limit of IL-2 detection is denoted by the horizontal dashed line. Data are representative of (A) four mice from two individual experiments or (B–E) pooled from at least two separate experiments with each point representing an individual mouse.

Effector differentiation of CD8⁺ T cells induces IL-2–dependent Treg expansion. (A) CD45.1⁺ OT-I or CD45.1⁺ gBT-I T cells were transferred to nTx or sham-Tx 11c.OVA mice treated with anti-CD25 or isotype control mAb. At the indicated time points, spleens were collected and the number of CD4⁺CD25⁺FoxP3⁺ Treg determined by FACS counting assay. (B) CD45.1⁺ OT-I or CD45.1⁺ gBT-I T cells were transferred to nTx 11c.OVA mice treated with anti–IL-2 (JES6.1 plus S4B6) or PBS, as indicated. Three days after transfer, spleens were collected and the number of CD4⁺CD25⁺FoxP3⁺ Treg determined by flow cytometry. Data are (A) mean ± SD (n = 4–7 per group) or (B) individual values pooled from two to three experiments.

Regulatory T cells restrain CD8⁺ expansion and effector differentiation. (A–C) CD45.1⁺ OT-I T cells were transferred to anti-CD25 or isotype-treated C57BL/6 mice immunized with OVA/QuilA at the time of transfer. At the indicated time points, inguinal LN (A) or spleens (B and C) were collected and OT-I number determined by a FACS counting assay and intracellular cytokine staining performed. (D) CD45.1⁺ OT-I T cells were transferred to anti-CD25 or isotype-treated C57BL/6 mice immunized with OVA/QuilA at the time of transfer and 21 d later. A further 7 d later, spleens were collected and the total number of OT-I cells determined using a FACS counting assay. (E–H) CD45.1⁺ OT-I T cells were transferred to nTx or sham-Tx 11c.OVA mice treated with either anti-CD25 or isotype-control mAb. At the indicated time points (E and F) or 3 d after transfer (G and H), spleens were collected and OT-I T-cell number determined by a flow-cytometric counting assay and intracellular cytokine staining performed. Data depict mean ± SEM or individual values for 4 to 10 mice for each time point (A–C) pooled from a minimum of two experiments, except inguinal LN 1 d posttransfer, where two mice from a single experiment are represented, and (D–H) four to six mice per group pooled from two to three individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Increased expansion and effector differentiation in the absence of Treg is IL-2–dependent. (A) CD45.1⁺ OT-I T cells (2 × 10⁶) were transferred to anti-CD25 or isotype-treated C57BL/6 mice immunized or not with OVA/QuilA at the time of transfer and additionally administered anti–IL-2 (JES6.1 plus S4B6) or PBS, as indicated. Seven days later, spleens were collected and the total number of OT-I T cells determined by FACS counting assay. (B) CD45.1⁺ OT-I T cells were transferred to anti-CD25 or isotype-treated nTx 11c.OVA mice administered either anti–IL-2 or PBS. Three days later, spleens were collected and the total number of OT-I T cells determined by FACS counting assay. (C and E) CD45.1⁺ OT-I T cells were transferred to DEREG or nontransgenic mice treated or not with DT, anti–IL-2, or PBS and immunized with QuilA at the time of transfer. Seven days later, spleens (C) were collected and the total number of OT-I T cells determined by FACS counting assay and serum IL-2 determined by ELISA (E). (D and F) CD45.1⁺ OT-I T cells were transferred to C57BL/6 mice treated or not with anti-FR4 and anti–IL-2 and immunized with OVA/QuilA at the time of transfer. Seven days later (D), spleens were collected and the total number of OT-I T cells determined by FACS counting assay and serum IL-2 determined by ELISA (F). (G) C57BL/6 mice were treated or not with anti-CD25 or anti-FR4. DEREG mice were treated with DT and 7 d later serum collected and IL-2 measured by ELISA. (H) CD45.1⁺ OT-I T cells (2 × 10⁴) were transferred to anti-CD25 or isotype-treated C57BL/6 mice administered either anti–IL-2 or PBS and immunized with OVA/QuilA at the time of transfer. Seven days later, spleens were collected and the total number of OT-I T cells determined by FACS counting assay. Data are pooled from at least two separate experiments and each datapoint portrays an individual mouse.