Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes (original) (raw)
Effect of IL-2 and IL-15 on the proliferation and function of antigen-activated CD8+ T cells. First, we compared the ability of IL-2 and IL-15 to support the proliferation and functional differentiation of antigen-stimulated CD8 T cells obtained from P14 mice. These mice express a transgene-encoded TCR specific for the immununodominant LCMV glycoprotein peptide, gp 33-41 (KAVYNFATM), presented in the context of H2-Db class I MHC molecules (13). P14 splenocytes were stimulated with LCMV gp33-41 synthetic peptide (hereafter referred to as gp33 peptide) and cultured for 2 days without cytokines, following which the cells were washed and cultured in varying concentrations of either IL-2 or IL-15. Cell proliferation was measured by BrdU incorporation, and viable cells were counted at 24-hour intervals throughout the 5-day period of cytokine treatment. In the absence of any cytokines, activated cells did not proliferate significantly, but rapidly decreased in numbers (Figure 1, a and b). In contrast, cells cultured either with IL-2 at all concentrations tested (1–100 ng/ml) or with IL-15 at 5 ng/ml or greater, proliferated equally well during the first 3 days of cytokine treatment. At days 4 and 5 the viable cell numbers in IL-2–treated cultures began to plateau, whereas IL-15–treated cultures continued to expand. These differential effects of IL-2 and IL-15 possibly were due to their respective pro- and antiapoptotic effects on CD8 T cells, as evidenced here by propidium iodide staining (Figure 1c) and observed previously in other model systems (8).
Effect of IL-2 and IL-15 on activated CD8 T cell proliferation and function. (a and b) All tested concentrations of IL-2 and IL-15 at 5 ng/ml or greater support cell proliferation. Cell proliferation was measured by viable cell counts (a) or BrdUincorporation (b) as described in Methods. In b, histograms of BrdU incorporation by CD8+ gated cells on day 3 of cytokine treatment are shown. Background staining obtained with isotype control is also shown for comparison. Similar results were seen for all treatment groups on each day for all 5 days tested (not shown). (c) IL-2 induces increased cell death compared with IL-15. Cell death was measured by flow cytometric analysis of non-gated cells after staining with propidium iodide (PI). Shown are dot plots after 3 days of cytokine treatment. (d) Both IL-2– and IL-15–treated cells acquire the ability for IFN-γ production. On day 7 following peptide stimulation and cytokine treatment, the cultures were restimulated with αCD3 for 6 hours, stained externally with αCD8 Cy 5, intracellularly with αIFN-γ Ab, and examined by flow cytometry. Less than 2% of unstimulated cells produced IFN-γ (not shown). (e) Peptide primed cells cultured with IL-2 at concentrations of 10 ng/ml or greater exhibit high levels of cytotoxicity, whereas cells cultured with IL-2 at or below 5 ng/ml and all tested concentrations of IL-15 exhibit greatly diminished levels of cytotoxicity. On day 7 after stimulation, cultures were tested for specific killing of gp33 peptide-pulsed EL-4 target cells by chromium-release assay.
Effector functions were evaluated 7 days after stimulation (5 days after cytokine treatment) by testing for the cells’ ability to produce IFN-γ and to mediate peptide-specific cytotoxicity. Both IL-2 and IL-15 supported IFN-γ production equally well (Figure 1d). However, while cells cultured in IL-2 at 10 ng/ml or greater were highly cytotoxic, cells cultured in IL-15 or IL-2 at doses of 5 ng/ml or less exhibited little cytotoxicity (Figure 1e). To determine if cells cultured in IL-15 acquired cytotoxic potential transiently, we also tested cytotoxicity 2 and 4 days after peptide activation. While cytotoxic potential progressively increased for cells cultured in high-dose IL-2, IL-15–treated cells did not exhibit comparable levels of cytotoxicity at any time point (not shown). Thus, a threshold dose of IL-2 appears to be necessary for differentiation of antigen-activated CD8 T cells into full-fledged effector cells, whereas any doses of IL-15 above 5 ng/ml and subthreshold doses of IL-2 support proliferation and IFN-γ production, but do not support differentiation into effector CTLs.
IL-2 and IL-15 induce distinct phenotypic changes in antigen-activated CD8+ T cells. Several studies suggest that compared with effector cells, memory cells exhibit reduced levels of cytotoxicity (19–23). Thus, we examined cells cultured in IL-2 and IL-15 for expression of phenotypic markers that are preferentially associated with naive, effector, or memory cells.
Until recently, there was no cell surface marker that could unambiguously differentiate between effector and memory CD8+ T cells. We have described recently T-GFP mice in which loss of GFP expression characterizes effector CTLs (12). Subsequently, Sallusto et al. have reported that a subset of memory T cells (called central memory cells) are characterized by lack of immediate effector function and retention of L-selectin and the chemo-kine receptor CCR-7 (24). Moreover, Harrington et al. have reported that the CD43 epitope recognized by the mAb, 1B11 is expressed on effector, but not memory CD8 T cells (23). Thus, we crossed P14 mice with T-GFP mice and examined the double-Tg CD8 T cells before and after peptide and cytokine treatment for expression of these markers as well as the common T cell activation markers CD69, CD25, and CD44.
Freshly isolated CD8+ T cells from the double-Tg mice expressed surface markers characteristic of naive T cells; that is, they did not express CD69, CD44, CD25, or the CD43 epitope 1B11, and were uniformly L-selectin– and GFP-positive (Figure 2a). When the P14XT-GFP splenocytes were stimulated in vitro with the gp33 peptide, CD8+ T cells developed a blastoid morphology and expressed high levels of all activation markers except the CD43 epitope 1B11, but retained GFP and L-selectin expression during the first 2 days of stimulation (Figure 2b).
To study the effect of cytokines, the peptide-activated cells were washed after 2 days of stimulation and cultured in high doses (20 ng/ml) of IL-2 or IL-15 for an additional 5 days before testing the phenotype. While cells cultured in IL-2 maintained the large blastoid size, IL-15–treated cells were smaller, as determined by their forward scatter profile (Figure 2c). Flow cytometric analysis also revealed that the cells cultured in IL-2 had uniformly lost GFP, L-selectin, and CCR-7 expression. However, they were strongly positive for the CD43 1B11 epitope. In contrast, cells maintained in IL-15 mostly retained GFP, L-selectin, and CCR-7 expression, but did not express the 1B11 epitope.
Appearance of the CD43 1B11 binding epitope on T cells requires modification of _O_-glycans by core 2 _N_-acetylglucosaminyltransferase (C2 GlcNAcT). C2 GlcNAcT activity is induced by T cell activation, but is downregulated in memory T cells, which return to expressing a core 1 _O_-glycan with increased sialylation (25). Because modification by C2 GlcNAcT is also required for P-selectin glycoprotein ligand–1 (PSGL-1) to bind P-selectin (26), we tested P-selectin binding activity after cytokine treatment using recombinant P-selectin Ig chimera (18). Strong P-selectin binding was seen only in the IL-2–treated cells. Naive cells, 2-day peptide-activated cells, and the IL-15–treated cells all failed to bind P-selectin.
Among the activation markers, the IL-2–treated cells maintained high levels of CD69, CD44, and CD25, while the IL-15–treated cells showed intermediate levels of expression, most notably for CD69 and CD25.
Taken together, the IL-2–stimulated peptide-primed CD8+ T cells lost GFP, L-selectin, and CCR7 expression, but expressed high levels of CD69, CD25, CD44, andCD43 1B11 epitope and functional PSGL-1. In contrast, the IL-15–treated cells retained GFP, L-selectin, CCR7, and CD44 expression, but downregulated CD69 and CD25 and did not bind 1B11 or P-selectin. These phenotypic differences suggest that while high-dose IL-2 differentiates activated CD8+ T cells into effector cells, IL-15 may drive them toward a memory phenotype that is reminiscent of central memory cells (24). Cells cultured in low-dose (5 ng/ml) IL-2 developed a phenotype similar to IL-15–treated cells (not shown).
Fully differentiated effector CTLs revert to a memory phenotype and function under the influence of IL-15. We also tested if IL-15 could convert differentiated effector cells into memory-like cells. For these studies, effector cells were generated in the presence of 20 ng/ml IL-2, and on day 7 the cells were washed and cultured with 20 ng/ml of either IL-2 or IL-15 for 5 more days before testing their phenotype and function. The 7-day-old effector cells cultured in either IL-2 or IL-15 remained L-selectin and GFP negative. However, effector cells that were switched to IL-15 downregulated CD69 and CD25 and had reduced levels of cytotoxicity compared with parallel cultures treated continuously with IL-2 (Figure 3). These results suggest that IL-15 can convert CD8+ T cells that had previously differentiated into effector CTLs to cells of memory phenotype and function, even though they differed from cells that were only exposed to IL-15 with regard to expression of homing molecules and GFP.
IL-15 reduces activation and cytotoxic function of fully differentiated effector CTLs. P14XT-GFP Tg splenocytes were stimulated with the gp33 peptide and cultured in 20 ng/ml IL-2 for 7 days. The cells were then washed and maintained in either IL-2 or IL-15 (20 ng/ml) for the next 5 days before testing for activation phenotype (a) and peptide-specific cytotoxicity (b). Overlay histograms of cells cultured in IL-2 (squares) and IL-15 (circles) are shown after gating for CD8 expression in a. Data for two sets of independent cell lines are shown in b.
Rapid induction of effector functions in CD8+ T cells cultured in IL-15 upon rechallenge. A hallmark of memory CD8+ T cells is their ability to proliferate and to acquire cytolytic activity rapidly after restimulation. Thus, we tested the proliferation, phenotype, and function of peptide-primed P14XT-GFP cells treated with IL-15 for 7 days and, subsequently, restimulated with αCD3 or gp33 for 2 days. Compared with unstimulated cells, the restimulated cells proliferated more vigorously (Figure 4a), rapidly lost GFP and L-selectin expression and upregulated CD69, CD25, and CD44 expression (Figure 4b, right and middle panels). This phenotype of restimulated cultures closely resembled that seen in parallel cultures continuously maintained in IL-2 (Figure 4b, left panel). Moreover, the restimulated cells had acquired the capacity to mediate a high degree of peptide-specific cytotoxicity compared with unstimulated cells within 2 days of stimulation (Figure 4c). Similar results were obtained when peptide lines generated with low doses of IL-2 (5 and 1 ng/ml) were restimulated (not shown). In contrast, antigenic stimulation of naive CD8+ cells did not induce CTL activity by 2 days of stimulation (not shown). Thus, cells generated under the influence of IL-15 or low doses of IL-2 are capable of rapid-recall responses in vitro.
TCR stimulation induces cells maintained in IL-15 to proliferate vigorously and to rapidly acquire effector phenotype and function. Tg splenocytes were stimulated with gp33 peptide, maintained in IL-15 for 7 days and then were restimulated with αCD3 for 48 hours or maintained in IL-15 without restimulation. Subsequently, both populations were tested for proliferation by thymidine incorporation (a), activation status (b), and peptide-specific cytotoxicity (c). In b the phenotype of cells maintained in parallel with a high dose of IL-2 (20 ng/ml) is also shown for comparison. Peptide-stimulated cells maintained in low doses of IL-2 (5 ng/ml or less), which resembled IL-15–treated cells in phenotype and function, also proliferated and rapidly acquired effector phenotype and cytotoxic function after restimulation (not shown).
Antigen-primed CD8 T cells treated with IL-15 and low-dose IL-2 are capable of long-term survival and mount rapid-recall responses in vivo. Cells differentiated with IL-2 and IL-15 were compared with each other and to in vivo–generated memory cells for their ability to mediate recall responses in adoptive transfer experiments. P14 mice that were backcrossed into the C57/BL6 background for over 10 generations (gift of Rafi Ahmed) were used as T cell donors. Groups of C57/BL6 mice were injected intravenously with 106 CD8+ T cells obtained from naive P14 mice or with peptide primed cells maintained in 20 ng/ml or 5 ng/ml IL-2, or 20 ng/ml IL-15, or kept without cell injection as a control. To generate memory cells in vivo, 2 days after transfer one group of mice injected with naive cells were infected with recombinant vaccinia virus encoding the LCMV glycoprotein (14). All mice were rested for 10 weeks and were then challenged intraperitoneally with recombinant Listeria monocytogenes encoding the LCMV gp33 epitope. On day 4 after challenge, PELs were harvested and tested for peptide-specific IFN-γ production and cytotoxicity (Figure 5). Control mice that were not adoptively transferred did not exhibit peptide-specific IFN-γ production or cytotoxicity. Thus, endogenous gp33 response was not significant on day 4 after infection, and we could attribute the response seen in other experimental groups to be due to the adoptively transferred donor cells. Mice transferred with naive TCR Tg cells showed small but detectable levels of IFN-γ production and cytotoxicity. In contrast, mice that were transferred with IL-15 or low-dose IL-2–treated cells exhibited a vigorous response in both IFN-γ production and cytotoxicity that was equivalent in magnitude to the response seen with in vivo–generated memory cells. Mice that received high-dose IL-2–treated cells showed an intermediate response. Thus, in vitro–activated CD8 T cells treated with IL-15 or low-dose IL-2 show the long-term survival and rapid-recall responses in vivo that are characteristic features of bona fide memory T cells.
Function of activated CD8+ T cells differentiated with IL-2 and IL-15 after adoptive transfer. P14 Tg splenocytes were stimulated with peptide for 2 days, washed, and cultured with a high (20 ng/ml; AT/IL-2 Hi) or low (5 ng/ml; AT/IL-2 Lo) dose of IL-2 or IL-15 (20 ng/ml; AT/IL-15) for 5 more days and infused intravenously into groups of C57/BL6 mice (106 cells per mouse; n = 3 mice per group). Mice were also infused with naive CD8+ T cells from P14 Tg mice (AT/Naive) or with PBS (No AT) as controls. To generate memory cells in vivo, some AT/Naive mice were infected with recombinant vaccinia virus encoding LCMV glycoprotein 2 days after transfer (AT/Naive/rVV). All mice were rested for 10 weeks, after which they were challenged with recombinant Listeria monocytogenes encoding the gp33 epitope; on day 4 after infection, PELs were tested for peptide-specific IFN-γ production (a) and cytotoxicity (b). Mean ± SD of all three experiments are shown in a, and results from one representative experiment (of three performed with similar results) are shown in b.