Essential role for interleukin-2 for CD4(+)CD25(+) T regulatory cell development during the neonatal period

Abstract

Although many aspects of CD4(+)CD25(+) T regulatory (T(reg)) cell development remain largely unknown, signaling through the IL-2R represents one feature for the production of T(reg) cells. Therefore, the present study was undertaken to further define early developmental steps in the production of T(reg) cells, including a more precise view on the role of interleukin (IL)-2 in this process. After adoptive transfer of wild-type T(reg) cells into neonatal IL-2Rbeta(-/-) mice, only a small fraction of donor T(reg) cells selectively seeded the lymph node (LN). These donor T(reg) cells underwent rapid and extensive IL-2-dependent proliferation, followed by subsequent trafficking to the spleen. Thus, IL-2 is essential for T(reg) cell proliferation in neonatal LN. The number and distribution of T(reg) cells in the periphery of normal neonatal mice closely paralleled that seen for IL-2Rbeta(-/-) mice that received T(reg) cells. However, for normal neonates, blockade of IL-2 decreased T(reg) cells in both the thymus and LN. Therefore, two steps of T(reg) cell development depend upon IL-2 in neonatal mice, thymus production, and subsequent expansion in the LN.

Figure 1.

Initial appearance of CD4⁺CD25⁺ T_reg cells in the lymph nodes of neonatal mice. Newborn C57BL/6 IL-2Rβ^−/− mice received purified GFP-CD4⁺CD25⁺ T cells (4–5 × 10⁵) and 6–20 d after transfer, spleen (SP), lymph node (LN), bone marrow (BM), and thymus cells from individual recipient mice were subjected to FACS analysis. (A) Dot plots for donor cells based on CD4 and GFP expression. The percent of cells expressing CD4 and/or GFP is indicated in appropriate quadrant. (B) The percent of donor GFP⁺ cells (left) or donor GFP⁺CD4⁺ of the total CD4⁺ cells (right) in the indicated organ. Data are mean ± SEM for three to five animals per day. (C) Dot plots for CD4 and CD8 expression for GFP⁺ donor cells. The percent of cells expressing CD4 or CD8 is indicated in the appropriate quadrant. (D) CD25 expression for CD4⁺ GFP⁺ donor cells. The percent of cells expressing CD25 is indicated in the upper right corner of each histogram. Data in A, C, and D are representative of three to five animals per day.

Figure 2.

CD4⁺CD25⁺ T_reg cells extensively proliferate in the lymph nodes of neonatal mice. Newborn C57BL/6 IL-2Rβ^−/− mice received purified CD45.1⁺ CFSE-labeled CD4⁺CD25⁺ T cells (4–5 × 10⁵) and 2–20 d after transfer, spleen (SP), and lymph node (LN) cells from individual recipient mice were subjected to FACS analysis. (A) Dot plots are shown for CD45.1 and CD4 expression (top) or CD25 and CFSE expression for CD45.1⁺CD4⁺ donor cells (bottom). The percent of donor CD4⁺ cells is indicated in the upper right corner of top dot plots. A total of 10⁵ or 10⁶ splenic cells were collected from 2- or 4-d-old mice, respectively. (B) Dot plots are shown for CD45.1 and CFSE expression. The percent of donor cells is indicated in the upper right corner of each dot plot. (C) Dot plots depicting CD4 and CD25 expression for CD45.1⁺ donor cells. The percent of cells expressing CD4 and CD25 of the gated populations is indicated in the upper right corner of the dot plots. Data in A and B are representative of two to five animals per day.

Figure 3.

Adoptive transfer of T_reg cells into IL-2Rβ^2/− mice largely follows the developmental scheme operative in normal mice. FACS analysis of lymph node and spleen cells was performed for 6–20-d-old mice, as indicated. The percent CD4- and CD25-positive cells (left) or the absolute number of CD4⁺CD25⁺ (right) in the (A) lymph node and (B) spleen. The absolute number of CD4⁺CD25⁺ T cells was calculated from the percent CD4⁺CD25⁺ cells in the lymph node and spleen times the number of cells in these organs. Data are mean ± SEM for three to seven mice per day per group. (C) CD69, CD62L, or CD103 expression for lymph node CD4⁺CD25⁺ T cells (n = 3–5 animals per group per day).

Figure 4.

The initial engraftment and proliferation of T_reg cells in neonatal lymph nodes requires IL-2. Neonatal IL-2Rβ^−/− mice were adoptively transferred with CD45.1⁺ CFSE-labeled CD4⁺CD25⁺ T cells (2 × 10⁵) and received either PBS or α-IL-2 mAb (100 μg) on the day of transfer and 3 d later. 6 d after transfer, lymph node cells from individual recipient mice were subjected to FACS analysis. (A) Histograms of donor (CD45.1) engraftment. (B) Dot plots for CD4 and CD25 expression after gating on CD45.1⁺ donor cells. (C) Histograms of proliferating donor CD4⁺CD25⁺ T cells based on CFSE staining. n = 3 per group.

Figure 5.

A Role for IL-2 in early development of CD4⁺CD25⁺ T_reg cells. B6 mice received either PBS (−) or α-IL2 mAb (+; 100 μg) on the day and 3 d after birth. 7 d after birth, lymph node cells from individual mice were subjected to FACS analysis. (A) The percent of CD4⁺CD25⁺ T cells. Data are mean ± SEM for 8–10 animals. (B) Dot plots of CD25 and GITR expression (left) and the absolute number of GITR⁺ cells for CD4⁺CD25⁺ cells (right). Data were mean ± SEM for three to four animals.

Figure 6.

IL-2 is required for thymic production of CD4⁺CD25⁺ T_reg cells. All mice received either PBS (−) or α-IL2 mAb (+; 100 μg) on the day and 3 d after birth. 7 d after birth, thymocytes from individual mice were subjected to FACS analysis with 250,000 events collected. In the data shown, the analysis of the indicated markers was performed after gating on CD4⁺CD8⁻ “single positive” thymocytes. (A) The percent of thymic CD4⁺CD25⁺ cells from B6 and Tg^−/− treated mice. Data are mean ± SEM for six to eight animals per group. *P < 0.05 compared with B6, and ^#P < 0.001 compared with Tg^−/− (one-way analysis of variance followed by Newman-Keuls Multiple Comparison). (B) Dot plots for CD25 and GITR expression for thymic CD4 single positive thymocytes from B6 mice (left) and the absolute number of GITR⁺ CD4 single positive thymocytes (right). Data are mean ± SEM for three to four animals per group.