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Evidence of a Novel IL-2/15R-Targeted Cytokine Involved in Homeostatic Proliferation of Memory CD8⁺ T Cells¹

Daisuke Kamimura^2,*, Naoko Ueda, Yukihisa Sawa, Shinji Hachida^*, Toru Atsumi, Takayuki Nakagawa, Shin-ichiro Sawa^*, Gui-Hua Jin^*, Haruhiko Suzuki, Katsuhiko Ishihara^*,, Masaaki Murakami^* and Toshio Hirano^3,*,,

^* Department of Molecular Oncology, Graduate School of Medicine and Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan; Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan; and Department of Immunology, Nagoya University School of Medicine, Nagoya, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The homeostasis of memory CD8⁺ T cells is regulated by cytokines. IL-15 is shown to promote the proliferation of memory CD8⁺ T cells, while IL-2 suppresses their division in vivo. This inhibitory effect of IL-2 appears to occur indirectly, through other cell populations including CD25⁺CD4⁺ T cells; however, the details of this mechanism remain unclear. In this study, we show that 1) both Ag-experienced and memory phenotype CD8⁺ T cells divided after the depletion of IL-2 in vivo; 2) this division occurred normally and CD44^highIL-2/15R^high CD8⁺ T cells generated after IL-2 depletion in IL-15 knockout (KO) and in IL-7-depleted IL-15 KO mice; 3) surprisingly, the blockade of IL-2/15R signaling in IL-2-depleted IL-15 KO mice completely abolished the division of memory CD8⁺ T cells, although the only cytokines known to act through IL-2/15R are IL-2 and IL-15; and 4) the expression of IL-2/15R molecules on memory CD8⁺ T cells was required for their division induced by IL-2 depletion. These results demonstrate that the depletion of IL-2 in vivo induced memory CD8⁺ T cell division by an IL-15-independent but by an IL-2/15R-dependent mechanism, suggesting the existence of a novel IL-2/15R-utilizing cytokine that acts directly on memory CD8⁺ T cells to promote cell division.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Long-lived memory CD8⁺ T cells expand more vigorously and produce effector molecules more rapidly than do naive cells upon re-encountering a pathogen and contribute to rapid clearance of the pathogen. It is thought that a constant pool of memory CD8⁺ T cells is maintained through a balance of slow basal proliferation and death (1, 2). This homeostasis of memory CD8⁺ T cells was shown to be established in the absence of MHC class I molecules (3). Subsequent studies revealed that cytokines, the IL-2 family in particular, are the principal mediators for the homeostasis of memory CD8⁺ T cells (4, 5, 6). At present, IL-2-family cytokines include IL-2, -4, -7, -9, -15, and -21 (7, 8). They have respective ligand-binding receptors (-chain), and share the common -chain (CD132, _c)⁴ for signal transduction. In addition, IL-2 and IL-15 require the IL-2/15R chain (CD122) for their signaling. IL-2R (CD25), IL-2/15R, and _c form a high-affinity receptor complex for IL-2 with K_a = 10^–11 M (7). Similarly, the IL-15R complex consists of IL-15R, IL-2/15R, and _c. But in contrast to IL-2R, IL-15R alone is capable of binding IL-15 with high affinity (K_a = 10^–11 M) in the absence of other receptor subunits (7).

We recently reported that endogenous IL-2 suppresses the division of memory CD8⁺ T cells in the homeostatic condition. A depletion of IL-2 leads to a significant increase in the basal homeostatic division of CD44^high memory phenotype CD8⁺ T cells, but not of CD44^low naive CD8⁺ T cells (9, 10). This inhibitory effect of IL-2 on memory phenotype CD8⁺ T cells in vivo appears to be indirect, given that an in vitro study revealed IL-2 alone stimulates the proliferation of memory CD8⁺ T cells and that it does not inhibit the IL-15-induced proliferation of nor induce apoptosis in these cells (10). A possible mediator cell for the in vivo IL-2 action is CD25⁺CD4⁺ regulatory T cells (10). Thus, the endogenous level of IL-2 negatively controls the homeostasis of memory CD8⁺ T cells in vivo. However, the detailed mechanism of IL-2 action in vivo remains obscure.

In contrast, the positive regulation of the cytokine-mediated homeostasis of memory CD8⁺ T cells has been revealed to be largely dependent on IL-15 (11, 12). For instance, IL-15 is a potent and selective growth factor for memory CD8⁺ T cells both in vitro and in vivo (13, 14, 15, 16), and numbers of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells are significantly decreased and their basal homeostatic proliferation is completely impaired in IL-15 knockout (KO) and IL-15R KO mice (17, 18, 19, 20, 21, 22, 23). Moreover, microbial components such as LPS and poly (I:C) indirectly induce the division of memory CD8⁺ T cells in vivo, in a process called "bystander proliferation," through IL-15 (24, 25). Interestingly, the expression of IL-15R on memory CD8⁺ T cells is dispensable for their bystander proliferation (24) and basal homeostatic proliferation (22, 23). A study using bone marrow-chimeric mice showed that IL-15R expression on radiosensitive hemopoietic cells is important for the proliferation of memory CD8⁺ T cells (22, 23, 24). Transpresentation of IL-15 by IL-15R-bearing non-T cells to CD44^high memory CD8⁺ T cells was observed in vitro (22, 26). It was also shown that memory CD8⁺ T cell proliferation after IL-15 injection in vivo requires the expression of IL-15R molecules on non-memory CD8⁺ T cells but requires that of IL-2/15R molecules by the memory CD8⁺ T cells (23), suggesting that the transpresentation of IL-15 to memory CD8⁺ T cells could occur in vivo. Another possibility that could account for the dispensability of IL-15R expression on CD8⁺ T cells is that the proliferation of memory CD8⁺ T cells may be mediated by an undefined growth factor released from IL-15R-bearing non-T cells, as proposed by Ma and colleagues (27).

We herein report that the MHC class I-independent division of memory CD8⁺ T cells induced by IL-2 depletion occurred normally even in the absence of IL-15. In addition, we provide evidence that this process was mediated by a putative novel IL-2/15R-utilizing cytokine, since treatment with anti-IL-2/15R Ab completely abrogated the anti-IL-2-mediated division of memory CD8⁺ T cells in IL-15 KO mice. Furthermore, we show that the expression of IL-2/15R molecules on memory CD8⁺ T cells was essential for action of the putative cytokine. These results demonstrate the existence of a previously undefined mechanism for the homeostasis of memory CD8⁺ T cells in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Retired C57BL/6 (B6) mice 5–7 mo of age were obtained from CLEA Japan (Osaka, Japan) or SLC (Shizouka, Japan). IL-15 KO, K^bD^b double KO, and B6.SJL (CD45.1)-congenic mice were obtained from Taconic Farms (Germantown, NY). IL-2 KO and B6.PL (Thy-1.1)-congenic mice were purchased from The Jackson Laboratory (Bar Harbor, ME). OT-1 TCR-transgenic and IL-7R KO mice were kindly provided by Drs. W. R. Heath (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) and K. Ikuta (Kyoto University, Kyoto, Japan), respectively. All strains used in this study were a B6 background. All of these mice except IL-2/15R KO mice were kept at the Institute of Experimental Animal Sciences at Osaka University Medical School (Osaka, Japan). IL-2/15R KO mice were kept at the Institute of Experimental Animal Sciences at Nagoya University Medical School (Nagoya, Japan).

mAb for flow cytometry and cell sorting

The mAbs used for flow cytometry and cell sorting were: PE-, PE-carbocyanin (Cy)5-, or APC-labeled anti-CD4 mAb (RM4-5); PE-Cy5- or APC-labeled anti-CD8 mAb (53-6.7); APC-labeled anti-CD25 mAb (PC61); FITC-, PE-Cy5-, or APC-labeled anti-CD44 mAb (IM7); biotinylated or APC-labeled anti-CD45.1 mAb (A20); biotinylated anti-CD45.2 mAb (104); APC-labeled anti-CD62L mAb (MEL-14); biotinylated anti-CD69 mAb (H1.2F3); APC-labeled Thy-1.1 (CD90.1) mAb (G7); PE-Cy5-labeled Thy-1.2 (CD90.2) mAb (53-2.1); biotinylated or PE-labeled anti-IL-2/15R (CD122) mAb (TM-1 and 5H4); biotinylated anti-IL-7R mAb (A7R34); and PE-labeled anti-V2 TCR mAb (B20.1). PE-Cy5- or APC-conjugated streptavidin was used for the biotinylated mAbs. These mAbs and streptavidin reagents were obtained from BD Biosciences (Mountain View, CA), eBioscience (San Diego, CA), BioLegend (San Diego, CA), and Caltag Laboratories (Burlingame, CA).

Adoptive transfer of donor T cells

Spleen and lymph nodes were harvested and homogenated by cell strainers (100 µm; BD Biosciences). The T cell-enriched fraction was obtained by passing the mixture of splenocytes and lymph node cells through nylon wool columns (Wako Biochemicals, Osaka, Japan and Polysciences, Warrington, PA). The T cell fraction was then sorted based on the CD4, CD8, CD44, and IL-2/15R levels using a cell sorter (MoFlo; DakoCytomation, Carpinteria, CA). The purity of the sorted populations was typically 97–99%. The purified populations were labeled with 5 µM CFSE (Invitrogen Life Technologies, Grand Island, NY) at 37°C. The CFSE-labeled donor cells (0.5–2 x 10⁶ cells/mouse) were transferred i.v. into nonirradiated mice. For Fig. 5B, the T cell-enriched fraction obtained from wild-type (WT) or IL-2 KO mice, which contained 3–5% of CD44^highIL-2/15R^high CD8 T cells, were used for donor cells.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. The anti-IL-2/15R mAb does not interfere with IL-7-mediated survival of CD44highIL-2/15Rhigh memory phenotype CD8+ T cells in vitro. CD44highIL-2/15Rhigh memory CD8+ T cells were cultured for 4 days without any cytokines (medium only) or in the presence of rIL-7 or IL-15 (100 ng/ml). Anti-IL-2/15R mAb (TM-1) or anti-IL-2 mAb (S4B6) was added at 100 µg/ml in some cultures. Living cells were detected as a 7AAD-negative population. The data represent the mean ± SD. Similar experiments were performed twice.

OT-1 CD8⁺ T cells (CD45.2) were transferred into B6.SJL (CD45.1) mice, followed by immunization i.p. and i.v. with extensively washed LPS-stimulated, OVA-pulsed bone marrow-derived dendritic cells. The immunized mice were then rested for at least 8 wk. Ag-specific OT-1 memory CD8⁺ T cells were sorted based on the expression of CD8⁺CD45.2⁺V2TCR⁺CD44^high. These cells were then labeled with CFSE and transferred into recipient mice.

Ab or poly(I:C) treatment

The hybridoma for anti-IL-2-neutralizing mAb (S4B6) was grown in hybridoma serum-free medium (Invitrogen Life Technologies). Anti-IL-2 mAb was then purified from the culture supernatant using a protein G column. The purified mAb was dialyzed against PBS and then sterilized by 0.2-µm pore filtration. Mice that received donor cells were given anti-IL-2 mAb i.p. five to seven times at 1 mg/mouse per day. Rat IgG (Sigma-Aldrich, St. Louis, MO) was used as a control Ab (9, 10). In some experiments, anti-IL-2R (PC61), anti-IL-2/15R (TM-1), anti-IL-7 (M25), anti-IL-7R (A7R34) mAb, or anti-c mAbs (4G3 and 3E12) was coinjected i.p. with anti-IL-2 mAb. These hybridomas except 4G3 and 3E12 (BD Pharmingen, San Diego, CA) were grown in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated FCS (Equitech-Bio, Kerrville, TX), and each mAb was purified as described above. The splenocytes and/or lymph node cells from treated animals were passed through nylon wool columns and the resulting T cell fraction was subjected to flow cytometry. The F(ab')₂ of anti-IL-2/15R mAb (TM-1) was prepared by pepsin digestion of the Ab at pH 4.0, followed by purification of the F(ab')₂ fraction using a Mono S cation exchange column or a gel filtration column (Takara Bio, Otsu, Japan). Contaminating whole IgG molecules in the F(ab')₂ preparation were below the detection limit by ELISA using an anti-rat Fc fragment-specific Ab (Valeant Pharmaceuticals, Costa Mesa, CA). Western blot analysis under nonreducing conditions using anti-rat IgG (H + L)-HRP (Zymed Laboratories, San Francisco, CA) was also performed. The intensity of the band corresponding to whole IgG (150 kDa) in the F(ab')₂ preparation was equivalent to a 1/1000 dilution of the original anti-IL-2/15R mAb. Poly(I:C) (Amersham Biosciences, Piscataway, NJ) was injected at 50–100 µg/mouse i.p. after donor T cell transfer.

In vivo BrdU labeling

Mice were given 0.8 mg/ml BrdU (Sigma-Aldrich) in their drinking water or it was administered i.p. at 1 mg/mouse during the anti-IL-2 treatment. BrdU staining for flow cytometry was performed using the BrdU Flow kit (BD Biosciences).

In vitro survival assay

CD44^highIL-2/15R^high CD8⁺ T cells were sorted and cultured at 1–2 x 10⁵ cells/well in RPMI 1640 medium plus 10% heat-inactivated FCS with or without murine IL-7 or IL-15 (100 ng/ml; PeproTech, Rocky Hill, NJ). In some wells, anti-IL-2/15R mAb (TM-1) or anti-IL-2 mAb (S4B6) was added at 100 µg/ml. After 4 days, the cells were stained with 7-aminoactinomicin D (7AAD, Via-Probe; BD Biosciences) and assayed for flow cytometry.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In vivo depletion of IL-2 selectively induced the homeostatic division of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells

Our previous reports revealed that IL-2 depletion induced the division of CD44^high memory phenotype CD8⁺ T cells, but not that of CD44^low naive CD8⁺ T cells (9, 10). To extend this finding, we first examined the effect of IL-2 depletion on the CD4⁺ as well as CD8⁺ T cell compartments. Memory CD8⁺ T cells can be distinguished from naive CD8⁺ T cells by the expression levels of several surface markers, such as CD44, IL-2/15R, and Ly6C (28). Similarly, the expression level of CD44 defines the naive and memory phenotype CD4⁺ T cell subsets. Naive CD4⁺ (CD44^low), memory phenotype CD4⁺ (CD44^high), naive CD8⁺ (CD44^lowIL-2/15R^low), or memory phenotype CD8⁺ (CD44^highIL-2/15R^high) T cells were sorted from the splenocytes and lymph node cells of retired mice, in which a large number of memory phenotype T cells accumulates (10, 29). These T cell populations bearing Thy-1.2 molecules were labeled with CFSE and transferred into nonirradiated Thy-1.1-congenic mice. The recipient mice were then given anti-IL-2-neutralizing mAb (S4B6). The Ab treatment in this experiment was designed to be a longer injection period (11 days) compared with our standard protocol (6–7 days) to perform a detailed examination whether CD4⁺ T cells have an ability to divide after anti-IL-2 treatment. Eleven days after the transfer, the division of donor T cells was detected by the dilution of CFSE. As shown in Fig. 1A, IL-2 depletion did not affect the division of CD44^low naive CD4 or CD44^low naive CD8 T cells. In contrast, the division of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells was significantly induced, as reported previously (9, 10). Consistent with this result, DNA synthesis, as judged by BrdU incorporation, was selectively increased by the anti-IL-2 treatment in the CD44^high fraction of CD8⁺ T cells compared with control IgG (rat IgG) treatment (Fig. 1B). The cell division and BrdU incorporation of CD44^high memory phenotype CD4⁺ T cells were not affected by the anti-IL-2 treatment protocols tested here (Fig. 1, A and B). Furthermore, the division of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells was also induced in IL-2 KO mice even without Ab treatment (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 1. IL-2 depletion selectively induces the division of CD44highIL-2/15Rhigh memory phenotype CD8+ T cells in vivo. A, CD44low or CD44highCD4+ T cells and CD44lowIL-2/15Rlow or CD44highIL-2/15Rhigh CD8+ T cells from mice expressing Thy-1.2 molecules were sorted and labeled with CFSE. The CFSE-labeled donor cells were transferred into nonirradiated syngeneic Thy-1.1 mice on day 0. Anti-IL-2 mAb (S4B6, 1 mg) or control Ab (rat IgG, 1 mg) was given i.p. on days 1, 3, 4, 5, 6, 7, and 8. CFSE levels were examined on day 11. Histograms shown are gated on the Thy-1.2-expressing donor population. B, Mice were given BrdU in their drinking water for 8 days. The anti-IL-2 treatment was performed for the first 6 days. The BrdU-positive population in the T cell compartments was examined. C, CFSE-labeled CD44highIL-2/15Rhigh CD8+ T cells were transferred into nonirradiated hosts on day 0. The anti-IL-2 treatment was conducted on days 1–5. Poly(I:C) was injected i.p. at 100 µg on days 1, 3, and 5. The CFSE levels and IL-2R, CD8, and TCR levels were examined on day 7. The plot of CFSE vs IL-2R was gated on lymphocyte (low side scatter but not dead cells) and TCR+ population and those of the others were gated on the lymphocyte population. The CFSE-negative population is derived from the host mouse. The results are representative of more than two to three independent experiments.

Taken together, these results suggest that the endogenous level of IL-2 selectively suppresses the homeostatic division of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells in vivo.

IL-2 depletion induced the division of Ag-experienced memory as well as CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells

CD44^highIL-2/15R^high memory-phenotype CD8⁺ T cells have many properties of memory cells (5, 31), but it is uncertain whether they have experienced Ag stimulation or not. To examine whether IL-2 also controls Ag-experienced memory CD8⁺ T cells, OVA-specific TCR-transgenic (OT-1) memory CD8⁺ T cells were generated (see Materials and Methods), CFSE-labeled, and transferred into Ag-inexperienced recipient mice. Similar to the CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells, the Ag-experienced OT-1 memory CD8⁺ T cells underwent several cell divisions after the anti-IL-2 treatment (Fig. 2). Given that both CD44^highIL-2/15R^high memory phenotype and Ag-experienced memory CD8⁺ T cells were found to be regulated by IL-2 in a similar way, we used CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells as donor cells in the following experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. IL-2 depletion induces Ag-experienced memory CD8+ T cells in vivo. OVA-specific OT-1 memory CD8+ T cells were generated in mice that received OT-1 CD8+ T cells and were subsequently immunized with OVA-loaded bone marrow-derived dendritic cells. CD44high OT-1 memory or CD44highIL-2/15Rhigh memory phenotype CD8+ T cells were labeled with CFSE and transferred into nonirradiated mice on day 0. The Ab treatment was performed on days 2–6. CFSE levels were examined on day 7. The CFSE-negative population is derived from the host mouse. The results are representative of three independent experiments.

It has been demonstrated that the maintenance of memory CD8⁺ T cells can be established independent of MHC class I molecules (3). To examine whether the division of memory CD8⁺ T cells induced by IL-2 depletion requires MHC class I molecules, CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells were transferred into MHC class I KO (K^bD^b double KO) mice. Consistent with our previous report, in which memory phenotype CD8⁺ T cells lacking ₂-microglobulin molecules could divide upon IL-2 depletion in ₂-microglobulin KO mice (9), donor CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells exhibited multiple divisions after IL-2 depletion in the recipients lacking MHC class I molecules (Fig. 3A). We also previously reported evidence suggesting that the inhibitory effect of IL-2 in vivo does not appear to be due to a direct action on memory phenotype CD8⁺ T cells (10). These observations led us to hypothesize that endogenous IL-2 suppresses the activity of a soluble factor, such as a cytokine, that triggers memory phenotype CD8⁺ T cell-specific proliferation in vivo. The most likely candidate for this agent was IL-15, because IL-15 is a potent growth factor for memory CD8⁺ T cells and currently known mechanisms for the Ag-independent division of these cells in vivo are largely dependent on IL-15 (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 27, 31). To test this hypothesis, CFSE-labeled CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells isolated from WT mice were transferred into IL-15 KO mice, and the host mice were then treated with anti-IL-2 mAb. Surprisingly, the division of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells was induced normally in the IL-15 KO hosts (Fig. 3B), even though we could not see any division of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells in IL-15 KO mice after poly(I:C) treatment as described previously (Fig. 3B) (25).

View larger version (21K): [in this window] [in a new window]   FIGURE 3. IL-2 depletion induces the division of CD44highIL-2/15Rhigh memory phenotype CD8+ T cells independently of IL-15. A, CFSE-labeled CD44highIL-2/15Rhigh CD8+ T cells sorted from WT mice (CD45.1) were transferred into nonirradiated WT or MHC class I KO (KbDb double KO, CD45.2) mice on day 0. The Ab treatment was performed on days 2–6. CFSE levels were examined on day 7. Histograms shown are gated on the CD45.1-expressing donor population. B, CFSE-labeled CD44highIL-2/15Rhigh CD8+ T cells were transferred into nonirradiated WT or IL-15 KO mice on day 0. Poly(I:C) was injected once at 50 µg/mouse, i.p. on day 2. The anti-IL-2 treatment was conducted on days 2–6. Control mice were given saline or rat IgG. CFSE levels were examined on day 7. C, WT or IL-15 KO mice were treated with anti-IL-2 mAb and BrdU for 5 days. Changes in the BrdU-positive population (top) and the endogenous CD44highIL-2/15Rhigh CD8+ T cell population (bottom) were examined on day 8. Plots shown were gated on the CD8-positive population. D, Anti-IL-7 and anti-IL-7R mAbs (M25 and A7R34, 0.5 mg each) were injected simultaneously with anti-IL-2 mAb for 5 days in IL-15 KO mice that have received CFSE-labeled CD44highIL-2/15Rhigh CD8+ T cells. B and D, The CFSE-negative population is derived from the host mouse. The results are representative of more than three to five independent experiments.

We next examined the involvement of IL-7 in this process, because the overexpression of IL-7 in mice overcomes the IL-15 dependency of CD44^high memory phenotype CD8⁺ T cells (34), and the irradiation-induced acute homeostatic proliferation of memory CD8⁺ T cells is halted when both IL-7 and IL-15 are suppressed (20, 35). Therefore, if IL-2 depletion up-regulates the production of IL-7, then CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells could proliferate even in the absence of IL-15. To examine this possibility, CFSE-labeled CD44^highIL-2/15R^high memory CD8⁺ T cells were transferred into IL-15 KO mice, and the mice were then treated with the combination of anti-IL-7 and anti-IL-7R mAbs to remove both IL-7 and IL-15 at the same time. They were then treated with anti-IL-2 mAb. As shown in Fig. 3D, inhibition of IL-7 and its signaling in IL-15 KO mice had no effect on the division of donor cells. B lymphopoiesis in the bone marrow was impaired in the anti-IL-7- plus anti-IL-7R mAb-treated mice, verifying the efficacy of these Abs (data not shown). In addition, the CD44^high memory phenotype CD8⁺ T cells from IL-7R KO mice divided in IL-15 KO mice after the anti-IL-2 treatment (data not shown). These results suggested that IL-2 negatively controlled the homeostasis of CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells in vivo in an IL-15-independent fashion by a mechanism that is distinct from MHC class I- or IL-7-dependent processes.

A putative novel IL-2/15R-utilizing cytokine mediates the division of memory CD8⁺ T cells

We demonstrated previously that anti-IL-2/15R treatment, which is known to block both IL-2 and IL-15 receptor-mediated signaling, diminishes the basal homeostatic division of memory phenotype CD8⁺ T cells in vivo (9). However, in the present study we found that CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells divided normally under the IL-2-depleted and IL-15-null condition (Fig. 3, B–D). Only IL-2 and IL-15 have been identified as IL-2/15R-utilizing cytokines. However, if there were a third IL-2/15R-utilizing cytokine or factor that induces the division of memory CD8⁺ T cells, it would explain the discrepancy noted above. Namely, under the IL-2-depleted, IL-15-null condition created by the anti-IL-2 treatment in IL-15 KO mice, the hypothesized third IL-2/15R-utilizing cytokine could induce the division of memory CD8⁺ T cells. To examine this hypothesis, IL-2-depleted IL-15 KO mice received CFSE-labeled CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells and were simultaneously treated with anti-IL-2/15R mAb. As expected, this treatment resulted in complete inhibition of the anti-IL-2-induced division ofCD44^highIL-2/15R^high memory phenotype CD8⁺ T cells in IL-15 KO mice (Fig. 4A). In contrast to anti-IL-2/15R, treatment with anti-IL-2R mAb had no such inhibitory effect, but rather intensified the efficacy of anti-IL-2 mAb alone in IL-15 KO mice (Fig. 4, A and C), indicating that the effect of the anti-IL-2/15R treatment was not simply due to a blockade of signaling from the remaining IL-2 molecules in the IL-2-depleted IL-15 KO mice. The first peak of CFSE signals from the donor IL-2/15R^high CD8⁺ T cells, which might be depleted by an anti-IL-2/15R complement reaction, was clearly detectable in the anti-IL-2/15R-treated mice (Fig. 4A), and the F(ab')₂ of anti-IL-2/15R mAb was also effective (Fig. 4B), excluding the possibility of the Fc-mediated depletion of donor cells. The F(ab')₂ of anti-IL-2/15R mAb was less efficacious than the intact one in terms of blocking anti-IL-2-induced division of memory CD8 T cells (Fig. 4B). It was reported that a half-life of F(ab')₂ in vivo is generally shorter than that of whole IgG (36, 37). We think this could be the case. In addition, when compared with the intact anti-IL-2/15R mAb, at least five times more F(ab')₂ was needed to block the binding of a biotinylated anti-IL-2/15R mAb on CD44^highIL-2/15R^high CD8⁺ T cells (data not shown), suggesting that removal of the Fc portion of Abs might also affect their avidity to Ags. As shown in Fig. 4C, the anti-IL-2/15R treatment also negated the emergence of the CD44^highIL-2/15R^high CD8⁺ T cell population induced by IL-2 depletion in IL-15 KO mice.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. A putative IL-2/15R-utilizing factor mediates the division of CD44highIL-2/15Rhigh memory phenotype CD8+ T cells induced by IL-2 depletion. A, CFSE-labeled CD44highIL-2/15Rhigh CD8+ T cells sorted out from WT mice were transferred into nonirradiated IL-15 KO mice on day 0. Treatment with anti-IL-2 mAb was performed simultaneously with anti-IL-2/15R mAb (TM-1, 0.03 mg) or anti-IL-2R mAb (PC61, 0.3 mg) on days 2–6. CFSE levels were examined on day 8. B, Nonirradiated IL-15 KO mice were transferred with CFSE-labeled CD44highIL-2/15Rhigh CD8+ T cells on day 0. The F(ab')2 of anti-IL-2/15R mAb (TM-1, 0.1 mg) was administered during the anti-IL-2 treatment on days 2–6. CFSE levels were examined on day 8. C, IL-15 KO mice were treated with control IgG alone (rat IgG, 1 mg), or with anti-IL-2/15R mAb (TM-1, 0.03 µg), or anti-IL-2R mAb (PC61, 0.5 mg) during the anti-IL-2 treatment for 5 days. The endogenous CD44highIL-2/15Rhigh population in CD8+ T cells was examined. Another anti-IL-2/15R mAb (5H4), which can bind to IL-2/15R molecules covered with TM-1, was used for flow cytometry. A and B, The CFSE-negative population is derived from the host mouse. The results are representative of more than three independent experiments.

Expression of IL-2/15R molecules on memory CD8⁺ T cells is essential for their division after IL-2 depletion

The results using anti-IL-2/15R mAb suggested the presence of the novel IL-2/15R-utilizing cytokine (Fig. 4), but they did not reveal which cell type(s) can respond to the putative cytokine. To investigate whether the putative IL-2/15R-utilizing cytokine acts directly on memory CD8⁺ T cells, CD44^low naïve or CD44^high memory phenotype CD8⁺ T cells were isolated from WT or IL-2/15R KO mice, labeled with CFSE, and transferred into IL-15 KO mice. These host mice were then treated with anti-IL-2 mAb. As shown in Fig. 6A, CD44^low naive CD8⁺ T cells were not affected by IL-2 depletion, but CD44^high memory phenotype CD8⁺ T cells from WT mice underwent several divisions as expected. However, the donor CD44^high memory phenotype CD8⁺ T cells lacking IL-2/15R molecules failed to divide in response to the anti-IL-2 treatment. This result indicates that the putative cytokine acts directly through the IL-2/15R molecules on memory CD8⁺ T cells to promote their cell division.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. The expression of IL-2/15R on memory CD8+ T cells is essential for the division of these cells after IL-2 depletion. A, CD44high memory phenotype CD8+ T cells were sorted from WT or IL-2/15R KO mice (CD45.2), then labeled with CFSE and transferred into nonirradiated IL-15 KO mice (CD45.1) on day 0. Treatment with anti-IL-2 mAb was performed on days 1–5. The CFSE labels were examined on day 7. Histograms shown are gated on the CD45.2-expressing donor population. B, T cell-enriched fraction from WT or IL-2 KO mice (Thy-1.2) within 3 wk old was labeled with CFSE and transferred into nonirradiated WT hosts (Thy-1.1) on day 0. Anti-IL-2 treatment was performed on days 1–7. CFSE labels were examined on day 8. Histograms shown are gated on the Thy-1.2-expressing donor CD8+ population. The results are representative of two independent experiments.

The putative IL-2/15R-targeting factor is a member of the IL-2 family

It is well known that every member of the IL-2-family cytokines shares the _c molecule as a receptor component (7, 8). Therefore, we examined whether the division of memory CD8⁺ T cell induced by IL-2 depletion could be inhibited by anti-_c blocking mAb. CFSE-labeled CD44^highIL-2/15R^high memory phenotype CD8⁺ T cells isolated from WT mice were transferred into IL-15KO hosts. The host mice were then treated with the combination of anti-_c blocking mAbs (4G3 and 3E12) during the anti-IL-2 treatment. As shown in Fig. 7, the division of donor cells in response to IL-2 depletion was totally abrogated when _c signaling was blocked. This result suggests that the putative IL-2/15R-utilizing cytokine shares the _c and belongs to the IL-2 family of cytokines.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. A blockade of the c signaling abrogates the division of memory CD8 T cells after IL-2 depletion. CFSE-labeled CD44highIL-2/15Rhigh CD8+ T cells from WT mice were transferred into nonirradiated IL-15 KO mice on day 0. Treatment with control IgG (rat IgG, 0.2 mg) or anti-c mAbs (4G3 and 3E12, 0.1 mg each) was performed during the anti-IL-2 treatment (days 1, 2, and 4–6). CFSE levels were examined on day 7. The CFSE-negative population is derived from the host mouse. The results are representative of two independent experiments.

	Discussion

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Several lines of evidence suggest that the putative factor is not IL-2 itself. First, it is reasonable that there is almost no IL-2 in the mice treated with anti-IL-2 mAb, because we hardly saw CD25^highCD4⁺ regulatory T cells and clearly detected an excess amount of the Ab molecules in serum in the mice (data not shown). Second, a blockade of IL-2/IL-15R or _c signaling was inhibitory, whereas that of IL-2R signaling was stimulatory for the cell division induced by anti-IL-2 treatment (Figs. 1D, 4, and 7). Third, there is a direct evidence that autocrine IL-2 is not required for the memory CD8⁺ T cell division by an adoptive transfer of the cells from IL-2KO mice (Fig. 6B). Finally, if IL-2 acts in a paracrine manner even after anti-IL-2 treatment, memory CD4⁺ T cell division must have been observed (Fig. 1).

Theze and colleagues (39) reported that a chemically synthesized 30-aa fragment of the N-terminal human IL-2 (p1–30) acts as an agonist for the IL-2/15R signaling). In addition, several alternative forms of mouse IL-2 were listed in the expressed sequence tags databases, although there were no N-terminal short fragments similar to the p1–30 in the databases and S4B6, the anti-IL-2 mAb we used, recognizes an N-terminal portion of IL-2 molecules (p26–45) that overlaps with the p1–30 (40). However, it might still be possible to speculate that in vivo IL-2 depletion by S4B6 increases the activity of the alternative forms that do not contain the S4B6 determinant (e.g., p1–26). We hypothesize that such natural variants might act as agonists for IL-2/15R-induced signaling on memory CD8⁺ T cells, especially if the concentration of such variants increases after IL-2 depletion in vivo. To examine this possibility, we performed RT-PCR experiments using splenic cDNA with or without the anti-IL-2 treatment by several primer sets for the murine IL-2 gene. However, we did not observe a consistent result showing that the transcripts for the alternative forms significantly increased after anti-IL-2 treatment. Additionally, we have not seen the existence of the short N-terminal fragments of the IL-2 mRNA (data not shown). Furthermore, we observed that the blockade of _c signaling abrogated the division of memory CD8⁺ T cells after IL-2 depletion in IL-15 KO mice (Fig. 6B). This result shows a sharp contrast to the report by Eckenberg et al. (39), since they demonstrated that IL-2/15R-mediated, but not _c-derived, signaling is critical for the action of p1–30. Therefore, we suggest that the putative cytokine described here is not the natural variant(s) of IL-2 molecules that are agonistic to the IL-2/15R signaling, similar to p1–30.

Since a deficiency of IL-15 or IL-15R in mice leads to significant impairment of the maintenance of CD44^highIL-2/15R^high memory CD8⁺ T cells (17, 18, 19), we hypothesize that the putative IL-2/15R-utilizing factor would act downstream of or in parallel to IL-15. In this regard, it is intriguing that the expression of IL-15R on memory CD8⁺ T cells is dispensable, whereas its expression on the other cells is shown to be important for their IL-15-mediated division in vivo (22, 23, 24). It has also been reported that IL-2/15R, another component for IL-15R complex, is required to be expressed on memory CD8⁺ T cells to respond to IL-15 injection (23). Transpresentation of IL-15 by the IL-15R-bearing cells to other IL-2/15R- and _c-expressing cells such as memory CD8⁺ T cells (IL-15R^low or IL-15R^negCD44^high) has been reported in vitro (22, 26), which could explain the dispensability of IL-15R and the necessity of IL-2/15R expression on memory CD8⁺ T cells in vivo. Additionally, another possibility proposed by Ma and colleagues (27) is that the proliferation of memory CD8⁺ T cells may be induced by an undefined growth factor secreted from IL-15R-bearing non-T cells after IL-15 stimulation. The in vivo function of the putative IL-2/15R-utilizing cytokine suggested here might be consistent with their idea, if IL-15 induces and if IL-2 suppresses secretion of the cytokine or expression of its receptor component(s).

Blattman et al. (41) recently reported that the injection of rIL-2 increases the proliferation of virus-specific resting memory CD8⁺ T cells in mice. The administration of exogenous IL-2 is also beneficial for the proliferation of activated CD8⁺ T cells in vivo (42). One interpretation for the inconsistency between our results (i.e., IL-2 depletion induces the division of CD44^highIL-2/15R^high memory CD8⁺ T cells shown in this study and in Refs. 9 and 10) and theirs is that IL-2 acts as a two-edged sword: the endogenous, relatively low concentration of IL-2 is inhibitory, but the exogenous IL-2 supplement could raise the concentration to levels that are much higher than in the normal in vivo state, thereby promoting the division of memory CD8⁺ T cells. In fact, a high concentration of IL-2 stimulates the proliferation of CD44^high memory phenotype CD8⁺ T cells in vitro (10). It is likely that the rIL-2 treatment would have a direct action on memory CD8⁺ T cells even in vivo. In contrast, our previous data strongly suggest that the inhibitory effect of IL-2 on the division of memory phenotype CD8⁺ T cells is the result of an indirect action through other cell populations, including CD25⁺CD4⁺ T cells (10). Therefore, these distinct mechanisms of IL-2 action, i.e., direct vs indirect, on memory CD8⁺ T cells could cause the different outcomes.

In summary, our present study demonstrates that IL-2 negatively regulates the homeostatic division of memory CD8⁺ T cells through an IL-15-independent mechanism. In addition, we provide evidence that this process is completely dependent on IL-2/15R molecules expressed on memory CD8⁺ T cells, suggesting the existence of a third IL-2/15R-utilizing cytokine that regulates the homeostasis of memory CD8⁺ T cells in vivo, although we understand the possibility that differential efficacies of different Abs that may influence the results might be retained along with multiple indirect effects of Abs on various cell types in vivo. Molecular identification of the putative IL-2/15R-utilizing cytokine, one of our current research efforts, is thus an issue of great importance for understanding the regulation of memory CD8⁺ T cell homeostasis.

	Acknowledgments

 
We appreciate Dr. Phillipa Marrack (National Jewish Medical and Research Center, Denver, CO) for providing the hybridomas and for a critical reading of this manuscript. We also thank Dr. Brian C. Schaefer (Uniformed Services University of the Health Sciences, Bethesda, MD) for a critical discussion and a critical reading of this manuscript. We are grateful to Drs. William R. Heath (Walter and Eliza Hall Institute of Medical Research) and Koichi Ikuta (Kyoto University) for providing OT-1 TCR-transgenic mice and IL-7R KO mice, respectively. We also thank Ryoko Masuda and Ayako Kubota for their excellent secretarial assistance.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, the Kanae Foundation, the Uehara Foundation, and the Osaka Foundation for the Promotion of Clinical Immunology.

² Current address: Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, 230-0045, Japan. E-mail address: kamimura{at}rcai.riken.jp

³ Address correspondence and reprint requests to Dr. Toshio Hirano, Department of Molecular Oncology (C7), Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan. E-mail address: hirano{at}molonc.med.osaka-u.ac.jp

⁴ Abbreviations used in this paper: _c, common -chain; Cy, carbocyanin; KO, knockout; 7AAD, 7-aminoactinomicin D; WT, wild type.

Received for publication May 17, 2004. Accepted for publication September 3, 2004.

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