CD25+CD4+ T cells contribute to the control of memory CD8+ T cells

Abstract

Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. In animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.

Figure 1

IL-2 does not inhibit the proliferation or survival of CD8+ memory T cells in response to IL-15 in vitro. T cells were isolated from C57BL/6 mice, stained with anti-CD8 and anti-IL-2Rβ or anti-CD44, and sorted to yield memory CD8+ T cell populations that were CD8+IL-2Rβ^high or CD8+CD44^high. (A) Memory CD8+ T cells (5 × 10⁵ per ml) were cultured with IL-2 and/or IL-15 (R & D Systems). Their proliferation was assayed on day 2 by incorporation of MTT (Sigma) measured by OD₅₇₀. (B) Memory CD8+ T cells (2 × 10⁵ per ml) were cultured with 10 ng/ml IL-2 and/or IL-15 and the percentages of apoptotic cells were measured by their light-scattering properties on a flow cytometer 2 and 3 days later.

Figure 2

Age-dependent decrease of CD25+CD4+ and increase of CD8+ memory T cells in mice. Peripheral blood lymphocytes were isolated from C57BL/6 mice (1, 3, and 12 months old; n = 6). The cells were stained with anti-CD4, anti-CD8, anti-CD25, and anti-IL-2Rβ and analyzed. Data shown are the means and SDs.

Figure 3

CD25+CD4+ T cells require IL-2 for survival in mice. Lymph node and spleen T cells were isolated from C57BL/6 mice that have been injected with 1 mg/day of anti-IL-2 or control rat IgG for 5 days. The cells were stained with anti-CD4, anti-CD8, anti-CD25, and anti-IL-2Rβ and analyzed. The numbers of CD25^highCD4+ T cells in spleens and lymph nodes were 2.2 × 10⁵ ± 1.0 × 10⁵ in mice treated with rIgG (n = 4) and 0.65 × 10⁵ ± 1.1 × 10⁵ in mice treated with anti-IL-2 (n = 3). Data were analyzed by Student's t test; P < 0.011.

Figure 4

Increase in the numbers of CD8+ memory T cells induced by removal of IL-2 depends on the presence of CD4+ T cells. C57BL/6, Thy1.2+ mice were depleted of CD4+ T cells by treatment with a depleting anti-CD4 antibody. These animals, and control mice, were injected with Thy1.1+ T cells and anti-IL-2 or control rat IgG. Six days later mice were killed and the numbers of Thy1.1+CD8+ T cells in the animals counted. The experiments included two mice in each group; the results shown are representative of three experiments.

Figure 5

CD25+CD4+ T cells reduce the number of dividing memory phenotype CD8+ T cells in animals. T cells were purified from C57BL/6 (experiment 1) or C57BL/6.PL (experiment 2) mice, stained with anti-CD4, anti-CD8, and anti-CD25, and sorted to yield populations that lacked (CD25−CD4+) or included (CD25+CD4+) CD25+CD4+ T cells. The cells were labeled with CFSE and transferred into RagKO (experiment 1) or sublethally irradiated (450Rad, experiment 6) recipients. Other details of the experiments are listed in Table 1. Four days after transfer, T cells were isolated, stained with anti-CD8, anti-IL-2Rβ, or anti-CD44 and anti-Thy1.1 (experiment 2), and analyzed. In experiment 1, the CFSE-negative peak, consisting almost entirely of filler T cells, is not shown. In experiment 2, cells analyzed were gated to include only Thy1.1+ T cells. Data from these and four other similar experiments (Table 1) were analyzed by Wilcoxon and Student's t tests. Both analyses revealed statistically predictable decreases in the numbers of dividing memory phenotype CD8+ T cells in mice containing CD25+CD4+ T cells versus mice without CD25+CD4+ T cells (P < 0.05).