Naive CD8+ T cells differentiate into protective memory-like cells after IL-2 anti IL-2 complex treatment in vivo

Abstract

An optimal CD8(+) T cell response requires signals from the T cell receptor (TCR), co-stimulatory molecules, and cytokines. In most cases, the relative contribution of these signals to CD8(+) T cell proliferation, accumulation, effector function, and differentiation to memory is unknown. Recent work (Boyman, O., M. Kovar, M.P. Rubinstein, C.D. Surh, and J. Sprent. 2006. Science. 311:1924-1927; Kamimura, D., Y. Sawa, M. Sato, E. Agung, T. Hirano, and M. Murakami. 2006. J. Immunol. 177:306-314) has shown that anti-interleukin (IL) 2 monoclonal antibodies that are neutralizing in vitro enhance the potency of IL-2 in vivo. We investigated the role of IL-2 signals in driving CD8(+) T cell proliferation in the absence of TCR stimulation by foreign antigen. IL-2 signals induced rapid activation of signal transducer and activator of transcription 5 in all CD8(+) T cells, both naive and memory phenotype, and promoted the differentiation of naive CD8(+) T cells into effector cells. IL-2-anti-IL-2 complexes induced proliferation of naive CD8(+) T cells in an environment with limited access to self-major histocompatibility complex (MHC) and when competition for self-MHC ligands was severe. After transfer into wild-type animals, IL-2-activated CD8(+) T cells attained and maintained a central memory phenotype and protected against lethal bacterial infection. IL-2-anti-IL-2 complex-driven memory-like CD8(+) T cells had incomplete cellular fitness compared with antigen-driven memory cells regarding homeostatic turnover and cytokine production. These results suggest that intense IL-2 signals, with limited contribution from the TCR, program the differentiation of protective memory-like CD8(+) cells but are insufficient to guarantee overall cellular fitness.

Figure 1.

IL-2–anti–IL-2 complex treatment induces rapid activation of STAT5 in naive CD8⁺ T cells. Mice were given a single injection of IL-2–anti–IL-2 complexes. At the indicated time points, splenocytes were fixed and permeabilized for intracellular staining of the tyrosine-phosphorylated form of STAT5. Mice for the zero time point received no injection. The data are gated on the CD8⁺ population, and the numbers represent the percentage of gated cells in each quadrant.

Figure 2.

IL-2–anti–IL-2 complexes induce proliferation of naive antigen-specific CD8⁺ T cells. 0.5 × 10⁶ purified CD44^lo CD122^lo naive OT-I cells were transferred into nonirradiated mice on day −1. The mice were given IL-2–anti–IL-2 complexes or control (rat IgG) on days 0 and 2, and the assays were performed on day 6. (A) Proliferation of donor cells was evaluated by CFSE dilution. (B) Absolute number of donor cells (left) and host CD8⁺ T cells (right) in the spleen. The data represent the mean + SEM (n = 3). ***, P < 0.001 versus rat IgG treatment. (C) Phenotypic changes of the donor cells. Rat IgG and IL-2–anti–IL-2 complex treatment are indicated with open and shaded histograms, respectively. (D) IFN-γ production by donor OT-I cells was assessed after 4 h of stimulation in vitro. Numbers represent the percentage of gated OT-I cells making IFN-γ. (E) Equal numbers (1.1 × 10⁶ cells) of naive OT-I cells and IL-2–anti–IL-2 complex–activated OT-I cells were transferred to naive animals. An in vivo CTL assay with OVA peptide–pulsed (CFSE^hi) and control (CFSE^lo) targets was performed the next day. The killing activity was examined 20 h later. Numbers represent the percentage of CFSE^hi and CFSE^lo targets remaining. The donor cells (A–D) and target cells (E) were distinguished from host populations by congenic markers.

Figure 3.

Fcγ receptors are dispensable for the action of IL-2–anti–IL-2 complexes. B6 mice and FcRγ^−/−FcγRII^−/− mice were treated with IL-2–anti–IL-2 complexes on days 0 and 2. The mice were bled before starting the treatment (pretreatment) and on day 5. (A) Phenotype of the CD8⁺ T cell population of the same animal in each group is shown. The data are gated on CD3⁺ CD8⁺ cells. Numbers represent the percentage of gated cells within the box. (B) Absolute numbers of total leukocytes, CD8⁺ T cells (CD3⁺ CD8⁺), and NK cells (NK1.1⁺ CD3^NEG) in the peripheral blood before and after (day 5) the treatment. Each column represents the mean + SEM (n = 3). *, P < 0.05; or **, P < 0.01 versus pretreatment.

Figure 4.

IL-2–anti–IL-2 complexes stimulate naive CD8⁺ T cells with limited TCR stimulation. Host mice that received CFSE-labeled naive OT-I cells 1 d earlier were treated with IL-2–anti–IL-2 complexes on days 0 and 2. CFSE levels of donor cells from the spleen were examined on day 6. The data are gated on CD3⁺ CD8⁺ congenically marked donor cells. (A) WT and MHC class I K^b−/−D^b−/− mice that were irradiated with 10 Gy and transplanted with syngeneic BM cells were used as host animals. (B) Absolute number of OT-I cells recovered from the spleen of animals in A is shown. The data represent the mean + SEM (n = 2). ***, P < 0.001 versus rat IgG treatment. (C) Nonirradiated OT-I/RAG-1^−/− mice were used as hosts.

Figure 5.

IL-2–anti–IL-2 complexes generate memory cells with a central memory phenotype. Memory OT-I cells generated by LM-OVA infection (75 d after infection; population A), IL-2–anti–IL-2 complex memory OT-I cells (12 d of treatment plus 76 d after transfer; population B), and host CD8⁺ T cells (population C) were examined for the expression of cell-surface markers. Contour plots are gated on CD8⁺ cells. Numbers represent the percentage of CD8⁺ cells within the box.

Figure 6.

IL-2–anti–IL-2 complex memory CD8⁺ T cells protect against bacterial infection. (A) Enumeration of IL-2–anti–IL-2 complex memory OT-I/RAG-1^−/− cells (9 d of treatment and 91 d after transfer; CD45.2⁺) in the spleen before and 3 d after challenge with 10⁵ CFU of LM-OVA. The data are gated on CD8⁺ cells. Numbers represent the percentage of CD8⁺ cells within the box. (B) Absolute numbers of IL-2–anti–IL-2 complex memory OT-I/RAG-1^−/− cells in the spleen before and after LM-OVA challenge. Data represent the mean + SEM (n = 3–4). ***, P < 0.001. (c) CFU of LM-OVA in the spleen and liver at 3 d after challenge of mice containing equal numbers of naive OT-I/RAG-1^−/− or IL-2–anti–IL-2 memory OT-I/RAG-1^−/− cells. Data represent the mean + SEM (n = 4–6). ***, P < 0.001 versus the OT-I/RAG-1^−/− group.

Figure 7.

IL-2–anti–IL-2 complex memory CD8⁺ T cells display incomplete cellular fitness. (A) Mice bearing LM-OVA antigen-driven memory OT-I cells at 68 d after infection or IL-2–anti–IL-2 complex memory OT-I cells after 81 d (12 d of treatment plus 69 d after transfer) were given BrdU in drinking water for 7 d. BrdU incorporation by the indicated subsets of memory CD8⁺ T cells in the spleen is shown. Numbers represent the percentage of gated cells that are BrdU positive. (B) BrdU-positive population of host CD44^hi CD122^hi CD8⁺ T cells and IL-2–anti–IL-2 complex memory OT-I/RAG-1^−/− cells after 61 d (9 d of treatment plus 52 d after transfer). The data are the mean + SEM (n = 3). *, P < 0.05. (C–F) Splenocytes containing memory OT-I cells generated by LM-OVA infection (62 d after infection) or IL-2 signals (12 d of treatment plus 63 d after transfer) were stimulated in vitro with OVA peptide (C and D) or PMA plus ionomycin (E and F). Intracellular cytokine production was examined 4 h later. Numbers in C and E represent the percentage of gated cells in each quadrant. (D and F) The mean fluorescent intensity (MFI) of cytokine staining is shown (mean + SEM; n = 3). The MFI was calculated within the population positive for the respective cytokine. * and **, P < 0.05 and 0.001, respectively, versus the complex-driven memory cells; ***, P < 0.001 versus LM-OVA memory cells.