IL-7 promotes the transition of CD4 effectors to persistent memory cells

Abstract

After transfer to adoptive hosts, in vitro-generated CD4 effectors can become long-lived memory cells, but the factors regulating this transition are unknown. We find that low doses of interleukin (IL) 7 enhance survival of effectors in vitro without driving their division. When in vitro-generated effectors are transferred to normal intact adoptive hosts, they survive and rapidly become small resting cells with a memory phenotype. CD4 effectors generated from wild-type versus IL-7 receptor-/- mice were transferred to adoptive hosts, including intact mice and those deficient in IL-7. In each case, the response to IL-7 was critical for good recovery of donor cells after 5-7 d. Recovery was also IL-7-dependent in Class II hosts where division was minimal. Blocking antibodies to IL-7 dramatically decreased short-term recovery of transferred effectors in vivo without affecting their division. These data indicate that IL-7 plays a critical role in promoting memory CD4 T cell generation by providing survival signals, which allow effectors to successfully become resting memory cells.

Figure 1.

IL-7 promotes the survival and long term recovery of Th1 and Th2 effectors in vitro. (a) Effect of IL-7 on effector recovery in vitro. Th1 and Th2 effectors were generated from purified naive CD4 T cells from AND TCR Tg mice as in previous papers (references 2, 8). 10⁵/ml Th1 (left) and Th2 (right) effector cells were stimulated with 0, 0.1, 0.5, 2, and 10 ng/ml of IL-7 for 1, 2, 3, 4, and 6 d. Live cell recoveries were determined by counting live, trypan blue–excluding cells at the times indicated. Results are the mean count from triplicate cultures with standard deviations. This experiment is representative of three separate experiments. (b) The effect of IL-7 on effector division and viability in vitro. Cell cycle analysis. Th1 and Th2 effector cells were generated as described in a. After 4 d, the effector cell populations were collected and stimulated with or without high (5 ng/ml) or low doses (0.5 ng/ml) of IL-7 for 3 d. Recovered cells were stained with PI after ethanol fixation. We determined the fractions of cells in G0 or G1 (not depicted), in S, G2, or M (% dividing), and cells that were dead (% dead). Only single cells were analyzed. Results are representative of five separate experiments. (c) IL-7 up-regulates Bcl-2 gene expression. Th2 effector cells were stimulated without or with high (5 ng/ml) or low (0.5 ng/ml) doses of IL-7 for 16 h. RNA was isolated and expression of mRNA for a panel of death genes was analyzed by RNase protection assay using the mouse apoptosis-related template probe set (BD Biosciences). Results were similar in Th1 effectors and in a repeat experiment. The fold change relative to the L32 housekeeping gene for Bcl-2, Bcl-xL, and RIP at different doses of IL-7 is shown.

Figure 2.

Effectors become rested effector/memory cells after transfer. (a) Phenotypic shifts after transfer to adoptive hosts. Th2 effectors generated from AND.Thy1.1 donors were analyzed for FSC and expression of CD25, CD44, CD62L, and Fas by staining and FACS^® analysis (effector row). Effectors were transferred to intact B6 mice. After 2 and 4 d, hosts were analyzed. Cells from the spleen and lymph node were recovered and stained, and the coexpression of CD4⁺ Thy1.1⁺ (donor cells) and the markers was determined. (b) Effect of IL-7 on IL-7Rα expression (in vitro). In vitro–generated Th2 effectors were incubated with no cytokine (none), and low (1 ng/ml) or high (10 ng/ml) doses of IL-7 for 2 d. The expression of IL-7R was determined by staining and FACS^® analysis. (c) Expression of IL-7Rα on CD4 T cell subsets. The expression of IL-7R was determined on fresh naive CD4 T cells (naive) and on in vitro–generated Th2 effectors (effector), and those same cells rested for 2 d in vitro (rested effector). Th2 effectors were also transferred to intact B6 mice and evaluated after 2 d (2 d in vivo). For memory cells, Th2 effectors were transferred to Class II KO hosts, and the resulting memory cells evaluated after 8 wk (memory). IL-7R expression was determined on each of these populations.

Figure 3.

Effector cells that lack IL-7 response or are transferred to IL-7–deficient hosts survive poorly. (a) Transfer of effectors deficient in IL-7R expression. In vitro–generated AND IL-7R^−/− Th2 effector cells (Thy1.2) and WT AND Th2 effector cells (GFP⁺, Thy1.2) were mixed in equal proportions and injected into ATXBM hosts (Thy1.1) (left). middle, Class II KO hosts. right, intact hosts. After 4–7 d, spleen and lymph node cells were recovered and the percentage of the donor cells that were WT (GFP⁺, Thy1.2) versus IL-7R^−/− (GFP⁻, Thy1.2) is shown. Data are the means of three mice per group, with standard deviation. Similar effects were seen in two replicate experiments. (b) Effectors divide less and survive less well in IL-7–deficient hosts (left). WT in vitro–generated Th2 effectors were CFSE-labeled and transferred to groups of hosts, including IL-7^−/−, IL-7R^−/−, and Rag-2 KO mice. The number of donor cells recovered were determined by gating on CD4-CYC and Thy1.1-PE. Donor cell recovery at day 5 from the experiment described in panel a is shown (left). Similar reduction in the recovery of donor cells in IL-7^−/− compared with IL-7R^−/− was seen in the other experiments and at days 2 and 7 and at 6 mo. Results are representative of three separate experiments. Bcl-2 expression in different hosts (right). In the same experimental design, Th2 effectors (Thy1.1) were transferred into IL-7^−/−, IL-7R^−/−, and Rag-2^−/− hosts. Cells were recovered from the pooled spleen and lymph node at day 4 after transfer. Donor cell Bcl-2 expression was determined by intracellular staining with Ab to Bcl-2. Isotype control Ab staining (not depicted) was below 10 mean index of fluorescence in cells recovered from each host.

Figure 4.

Antibodies to IL-7 block recovery of transferred effectors without affecting division. In vitro–generated Th2 effector cells (Thy1.1) were adoptively transferred into Thy1.2 hosts including: intact B6 and Class II^−/− (Class II KO). (a) Blocking with anti–IL-7 in different hosts. Experimental groups were injected i.p. daily with anti–IL-7 (0.5 mg/mouse/d) from day −1, control groups were treated the same way, but with isotype-matched antibody. Results represent the mean donor cell recovery from individual animals (three to five mice per group), with standard deviations. Similar results were seen in a duplicate experiment and in other hosts, including RAG^−/− and ATXBM hosts (not depicted). (b) CFSE-labeled Th2 effector cells (Thy1.1) were adoptively transferred into intact and Class II^−/− (Class II KO) hosts. Experimental groups were injected i.p. with anti–IL-7 (0.5 mg/mouse/d) from day −1, control groups were treated the same way, but with isotype-matched antibody. After 3 d, CFSE staining of donor Thy1.1⁺ cells was assessed. Similar results were seen in a repeat experiment. No difference in CFSE profile between anti–IL-7–treated and isotype-treated hosts was seen after 5 d (not depicted).