IL-15-induced human DC efficiently prime melanoma-specific naive CD8+ T cells to differentiate into CTL

Authors

  • Peter Dubsky,

    1. Baylor Institute for Immunology Research, Baylor NIAID Cooperative Center for Translational Research on Human Immunology and Biodefense, Dallas, USA
    2. Medical University of Vienna, Department of Surgery, Vienna, Austria
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  • Hiroaki Saito,

    1. Baylor Institute for Immunology Research, Baylor NIAID Cooperative Center for Translational Research on Human Immunology and Biodefense, Dallas, USA
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  • Marylene Leogier,

    1. Baylor Institute for Immunology Research, Baylor NIAID Cooperative Center for Translational Research on Human Immunology and Biodefense, Dallas, USA
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  • Carole Dantin,

    1. Baylor Institute for Immunology Research, Baylor NIAID Cooperative Center for Translational Research on Human Immunology and Biodefense, Dallas, USA
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  • John E. Connolly,

    1. Baylor Institute for Immunology Research, Baylor NIAID Cooperative Center for Translational Research on Human Immunology and Biodefense, Dallas, USA
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  • Jacques Banchereau,

    1. Baylor Institute for Immunology Research, Baylor NIAID Cooperative Center for Translational Research on Human Immunology and Biodefense, Dallas, USA
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  • A. Karolina Palucka

    Corresponding author
    1. Baylor Institute for Immunology Research, Baylor NIAID Cooperative Center for Translational Research on Human Immunology and Biodefense, Dallas, USA
    • BIIR, 3434 Live Oak, Dallas, 75204 TX, USA, Fax: +1-214-8204813
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Abstract

Monocytes differentiate into dendritic cells (DC) in response to GM-CSF combined with other cytokines including IL-4 and IL-15. Here, we show that IL15-DC are efficient in priming naive CD8+ T cells to differentiate into melanoma antigen-specific cytotoxic T lymphocytes (CTL). While both melanoma peptide-pulsed IL15-DC and IL4-DC expand high-precursor frequency MART-1-specific CD8+ T cells after two stimulations in vitro, IL15-DC require much lower peptide concentration for priming. IL15-DC are more efficient in expanding gp100-specific CD8+ T cells and can expand CD8+ T cells specific for Tyrosinase and MAGE-3. CTL primed by IL15-DC are superior in their function as demonstrated by (i) higher IFN-γ secretion, (ii) higher expression of Granzyme B and Perforin, and (iii) higher killing of allogeneic melanoma cell lines, most particularly the HLA-A*0201+ Sk-Mel-24 melanoma cells that are resistant to killing by CD8+ T cells primed with IL4-DC. Supernatants of the sonicated cells demonstrate unique expression of IL-1, IL-8 and IL-15. Therefore, membrane-bound IL-15 might contribute to enhanced priming by IL15-DC. Thus, IL-15 induces myeloid DC that are efficient in priming and maturation of melanoma antigen-specific CTL.

Abbreviation:
actIL4/15-DC:

activated IL4/15-DC

Introduction

Cancer vaccines aim at inducing (i) tumor-specific effectors, able to reduce/eliminate the tumor mass, and (ii) long-lasting tumor-specific memory T cells, able to control tumor relapse. Owing to their capacity to induce and regulate T cell immunity, DC are increasingly used as adjuvants for vaccination in cancer 1, 2. While their safety and immunogenicity has been proven in early clinical trials 2, the next challenge is to distinguish which of the many subsets of human DC 3, 4 will make the “optimal” vaccine. The goal is to identify a cell that can efficiently expand rare tumor antigen-specific T cells and induce their maturation into specific CTL able to eliminate tumor cells.

While GM-CSF and possibly Flt-3L activate monocytes, other cytokines skew their differentiation into distinct DC subsets. For example, when activated monocytes encounter IL-4, they yield IL4-DC 5, 6. Upon encounter with IFN-α or TNF, activated monocytes differentiate into IFN-DC 79 or TNF-DC 10, respectively. Each DC subset displays common as well as unique biological functions determined by a distinctive combination of cell surface molecules and cytokines. In the human, this is best exemplified by interstitial DC and Langerhans cells where both subsets prime allogeneic naive T cells, while they differ in their capacity to activate naive B cells 11. They also differ in the pattern of cytokines they secrete, as well as in their enzymatic activity 11.

Administration of CD34-hematopoietic progenitor cell-derived DC (CD34-DC), loaded with keyhole limpet hemocyanin, Flu matrix peptide and melanoma peptides in HLA-A*0201+ patients with metastatic melanoma led to enhanced melanoma-specific CD8+ T cell immunity as measured by (i) IFN-γ ELISPOT 12, (ii) frequency of melanoma tetramer-binding CD8+ T cells, and (iii) killing of melanoma cells 13. The value of this vaccine might be related to its composite nature as it consists of at least two DC subsets and most particularly Langerhans cells which have not been assessed in any other clinical trial to date. In this context, we reported that IL-15 together with GM-CSF induces monocytes to differentiate into cells with properties of Langerhans cells (IL15-DC) and the preferential ability to enhance CD8+ T cell proliferation 14. We show here the efficiency of these cells to prime naive CD8+ T cells to differentiate into melanoma-specific CTL.

Results

IL15-DC induce strong proliferation of CD8+ T cells

Monocytes enriched by adherence and cultured, under clinical-grade conditions, for 3 days in the presence of 5% autologous serum and GM-CSF with either IL-4 (IL4-DC) or IL-15 (IL15-DC) yield comparable numbers of cells with the phenotype and morphology of DC (Fig. 1).

Figure 1.

GM-CSF in combination with IL-4 or IL-15 yields dendritic cells. Monocytes enriched by adherence were cultured for 3 days in the presence of GM-CSF + IL-4 or GM-CSF + IL-15, and subsequently activated by LPS (10 ng/mL). Giemsa staining of cytospun cells shows DC morphology before (top panels) and after LPS activation (bottom panels). Left panels show IL4-DC and right panels show IL15-DC.

When compared to IL4-DC, IL15-DC express higher levels of CD14 and CD80, and lower levels of CD1b/c, CD209 and CD86 (Fig. 2a, Table 1). IL15-DC uniquely express Langerin, a surface marker of Langerhans cells (not shown and 14). Activation with LPS induces a ∼10-fold increase in MHC class I and class II, CD80 and CD86 expression (Fig. 2b, Table 1). Activated (act)IL15-DC still express more CD80 and less CD86 than actIL4-DC.

Figure 2.

GM-CSF in combination with IL-4 or IL-15 skews monocyte differentiation into distinct DC subsets. Monocytes enriched by adherence were cultured for 3 days in the presence of GM-CSF + IL-4 or GM-CSF + IL-15, and subsequently activated by LPS (10 ng/mL). (a) Phenotype analysis by flow cytometry prior to LPS activation. Isotype control and specific staining with the indicated mAb. (b) Phenotype of DC subsets activated by LPS.

Table 1. Phenotype of IL4-DC and IL15-DC
IL4-DCIL15-DC
  1. a) The average MFI and the mean percentage of positive cells with the respective standard deviations (SD) of n independent experiments.

  2. b) The levels of statistical significance in a paired, two sided t-test.

Day 3 (immature)MFI ± SDa% positive cells ± SDMFI ± SD% positive cells ± SD
CD14; p <0.0001b(n = 10)7 ± 23 ± 1593 ± 6375 ± 8
CD209; p <0.002(n = 7)191 ± 2577 ± 473 ± 3136 ± 6
CD1b/c; p <0.002(n = 10)188 ± 2891 ± 556 ± 2631 ± 5
CD80; p <0.04(n = 6)14 ± 57 ± 362 ± 1525 ± 13
CD86; p <0.04(n = 7)140 ± 1680 ± 841 ± 871 ± 10
Day 4 (activated)MFI ± SD% positive cells ± SDMFI ± SD% positive cells ± SD
CD83; p <0.002(n = 5)53 ± 993 ± 632 ± 1136 ± 9
CD80; p <0.002(n = 5)191 ± 77>98%251 ± 67>98%
CD86; p <0.002(n = 5)223 ± 57>98%150 ± 49>98%

In cultures with allogeneic peripheral blood lymphocytes (PBL), IL15-DC yielded a cell population enriched in CD8+ T cells when compared to cultures with IL4-DC (Fig. 3a) (48 ± 8% with IL15-DC vs. 36 ± 18% with IL4-DC; n = 4, p <0.04; Fig. 3c). The overall cell recovery at the end of the culture was somewhat higher in cultures with IL15-DC ((1.86 ± 0.33) × 106 with IL15-DC vs. (1.56 ± 0.33) × 106 with IL4-DC; p = 0.02; Fig. 3d). A greater proportion of CD8+ T cells underwent cell divisions in response to IL15-DC than to IL4-DC (Fig. 3b). In transwell experiments where IL15-DC were added to the upper well, while lymphocytes were directly stimulated with IL4-DC in the lower well, IL15-DC were not able to enhance the CD8+ T cell proliferation induced by the IL4-DC (n = 3; p = 0.7; Fig. 3). Thus, the stimulatory effect on CD8+ T cells was not due to the release of soluble factors and/or to a carryover of IL-15 from the DC culture medium.

Figure 3.

IL15-DC induce strong CD8+ T cell proliferation among allogeneic PBL. Proliferation of PBL at 4 days after restimulation. (a) Expansion CD8+ T cells in cultures with IL15-DC (upper), but not with IL4-DC (lower) nor when IL15-DC were added to the upper well of a transwell system (middle) while PBL + IL4-DC were in the lower well. (b) Co-cultures stimulated with either DC subset for 7 days were harvested and stained with CFSE before restimulation. CFSE dilution in histograms gated on CD8+ T cells; a larger number of cells underwent five cell divisions in IL15-DC-stimulated PBL (upper). IL15-DC in the upper well did not enhance the CD8+ T cell proliferation in IL4-DC cultures (middle and lower). (c) Four paired observations from independent experiments show a statistically significant increase in CD8+ T cells in IL15-DC cultures in comparison to IL4-DC-stimulated PBL (paired t-test; upper). IL15-DC added in the upper well of a transwell system do not enhance CD8+ T cell percentages (lower). (d) Viable cell numbers (Trypan blue count) recovered from cultures with IL4-DC or IL15-DC; data from three independent experiments with cells from different donors.

IL15-DC skew naive CD8+ T cells towards effectors secreting type I cytokines

To determine whether the two DC subsets differentially polarize CD8+ T cells, naive allogeneic CD8+ T cells were stimulated with IL4- or IL15-DC for 7 days and restimulated with the relevant DC for 4 days. As shown in Fig. 4, a higher percentage of T cells acquired a CCR7 CD45RA effector memory phenotype in cultures with IL15-DC when compared to IL4-DC (Fig. 4a, d; n = 5, p <0.003). Accordingly, IL15-DC-stimulated cultures displayed a lower percentage of CD8+ T cells that retain a naive phenotype (Fig. 4a, d; CCR7+ CD45RA+; n = 5, p <0.03). Intracellular staining and flow cytometry further demonstrated that IL15-DC were more efficient than IL4-DC at inducing T cell differentiation as measured by the expression of Granzyme B (Fig. 4b, e; n = 4, p <0.01) and Perforin (Fig. 4b, e; n = 4, p <0.03). IFN-γ was expressed by T cells stimulated with either DC subset (Fig. 4c). Yet, IL15-DC caused a higher percentage of CD8+ T cells (Fig. 4a, f; n = 3, p <0.05) to express more IFN-γ [mean fluorescence intensity (MFI); Fig. 4c; 28.4 in IL4-DC vs. 85.8 in IL15-DC]. Conversely, CD8+ T cells cultured with IL15-DC expressed lower amounts of IL-4 (Fig. 4c; n = 3; p <0.02) than those stimulated with IL4-DC. These differences reflected enhanced T cell differentiation in cultures with IL15-DC rather than simple expansion, as the overall recovery of T cells in the end of the culture was not significantly different ((1.2 ± 0.37) × 106; range 0.4 × 106–1.1 × 106 with IL15-DC vs. (0.65 ± 0.17) × 106; range 0.55 × 106–2.2 × 106 with IL4-DC; n = 4, p = 0.1).

Figure 4.

IL15-DC skew naive CD8+ T cell differentiation towards effectors secreting Tc1-type cytokines. (a) IL15-DC (right) induce a higher percentage of effector memory CD8+ T cells, as shown by the loss of CCR7 and CD45RA, than IL4-DC (left). (b) Intracellular staining and flow cytometry reveal higher percentages of Granzyme B- and Perforin-positive cells in IL15-DC-stimulated cultures (right) than after stimulation with IL4-DC (left). (c) IL15-DC-stimulated CD8+ T cells (right) show a higher percentage of IFN-γ+ cells and the plots indicate a higher MFI than for IL4-DC (left). Conversely, IL4-DC induced more IL-4. CD8+ T cells were restimulated with PMA and ionomycin for 4 h. (d–f) Paired observations from independent experiments. Paired, two-tailed t-test.

LPS activation of IL4-DC did not compensate for their differential capacity to induce naive CD8+ T cell proliferation and turn on CD45RO expression (Fig. 5a; n = 3; p <0.04). Indeed, actIL15-DC were still more efficient than actIL4-DC at inducing the expression of Granzyme B and Perforin (Fig. 5b). Finally, actIL15-DC induced more IFN-γ than actIL4-DC (Fig. 5c). The percentage of cells expressing IL-4, IL-10 or IL-13 was below 1% in both conditions (n = 2; not shown). Thus, IL15-DC are efficient activators of naive CD8+ T cell growth and differentiation.

Figure 5.

LPS activation does not compensate for the differential capacity of DC subsets to induce naive CD8+ T cell proliferation, memory phenotype and expression of effector molecules and IFN-γ. (a) CFSE labeling of naive CD8 T cells combined with CD45RO staining after 7 days of stimulation with either actIL4-DC or actIL15-DC. The dot plot on the right, demonstrating cultures stimulated with actIL15-DC, depicts a higher percentage of cells to acquire CD45RO and increased proliferation. (b) Perforin and Granzyme B as demonstrated by intracellular staining are expressed at a higher amount in actIL15-DC-stimulated cultures. (c) CFSE-labeled naive CD8+ T cells were stimulated with PMA/ionomycin after 7 days of co-culture with actIL4-DC (left plot) or actIL15-DC (right plot). Intracellular staining for IFN-γ and flow cytometry shows that cells begin to express IFN-γ after approximately three divisions. actIL15-DC prompted more cells to proliferate than actIL4-DC, resulting in a higher percentage of these cells to express IFN-γ.

IL15-DC efficiently prime MAGE-3- and Tyrosinase-specific CD8+ T cells

A most desired property for a DC vaccine directed against cancer is the ability to efficiently prime naive, tumor-associated antigen-specific T cells into potent CTL. Naive CD8+ T cells from HLA-A*0201+ healthy volunteers were thus exposed to actIL4-DC or actIL15-DC, pulsed with four peptides derived from melanoma differentiation antigens: MART-1, gp100, Tyrosinase and/or MAGE-3. On day 7, T cells were restimulated with the relevant peptide-pulsed DC for five additional days. Differentiation of naive CD8+ T cells was measured by (i) tetramer binding and (ii) killing of melanoma antigen-expressing cells.

Both actIL4-DC and actIL15-DC induced expansion of MART-1-specific T cells (Fig. 6a, b; n = 13, p = 0.8; Table 2). However, actIL15-DC were more efficient than actIL4-DC at inducing the expansion of gp100-specific CD8+ T cells (Fig. 6a, b; n = 12, p <0.001; Table 2). The expansion of gp100-specific T cells in cultures with actIL15-DC was observed in 11/13 experiments performed with cells from six healthy volunteers. The two experiments that did not yield gp100-specific T cells were performed using cells from the same donor from which they could be expanded in two other experiments (Table 2). These results suggest the low frequency of gp100-specific naive CD8+ T cells in the blood of this individual. gp100-specific CD8+ T cells reached a maximum of 4.85% of the total CD8+ T cells primed with IL15-DC (mean 1.8 ± 1.6%). In contrast, actIL4-DC were able to induce gp100-specific CD8+ T cell growth (>0.1%) in 5/13 experiments reaching a maximum of 1.38% (mean 0.26 ± 0.42%) (Table 2).

Figure 6.

IL15-DC efficiently prime MART-1-, gp100-, MAGE-3- and Tyrosinase-specific CD8+ T cells from healthy volunteers. Autologous naive HLA-A*0201+ CD8+ T cells from healthy volunteers were cultured with actIL4- or actIL15-DC, pulsed with four melanoma peptides (10 µg/mL): MART-1, gp100, Tyrosinase, MAGE-3. On day 7, T cells were restimulated with the relevant peptide-pulsed DC for five additional days. (a) Melanoma tetramer binding in cultures with actIL4-DC (upper) and actIL15-DC. (b) Paired observations from each experiment (n = 13) plotted for the four specific peptides used for priming. In 13 experiments with cells from seven healthy volunteers, MART-1-, Tyrosinase- and MAGE-3-specific tetramer binding could be analyzed; in 12 experiments with cells from six healthy volunteers, gp100-specific staining could be analyzed. Wilcoxon matched pairs test shows significantly higher percentages of gp100-, MAGE-3- and Tyrosinase-specific CD8+ T cells in actIL15-DC- than in actIL4-DC-stimulated cultures. (c) Kinetics of tetramer binding at day 7 before restimulation, at day 10 and day 12. One representative experiment of three. (d) Viable cell numbers (Trypan blue count) recovered from cultures with IL4-DC or IL15-DC.

Table 2. Percentage of tetramer-specific CD8+ T cells elicited by either actIL4-DC or actIL15-DC
Donor/ExpaMART-1p = 0.8cgp100p = 0.001MAGE-3p = 0.037Tyrosinasep = 0.04
aIL4DCaIL15DCaIL4DCaIL15DCaIL4DCaIL15DCaIL4DCaIL15DC
  1. a) Autologous naive HLA-A*0201+ CD8+ T cells from six different healthy volunteers were cultured with actIL4-DC or actIL15-DC, pulsed with four melanoma peptides (10 µg/mL): MART-1, gp100, Tyrosinase, MAGE-3. See also Fig. 6.

  2. b) Melanoma tetramer binding (percentage of positive CD8+ T cells) in cultures with actIL4-DC and actIL15-DC. All experiments with the four melanoma peptides are shown for each donor.

  3. c) Wilcoxon matched pairs test.

#1 Exp 123.25b6.220.024.850.040.130.010.55
#1 Exp 27.362.500.012.980.040.040.022.47
#2 Exp 111.767.550.221.030.200.700.090.25
#3 Exp 18.5712.260.241.070.071.010.010.07
#3 Exp 210.7010.840.293.790.030.030.000.00
#4 Exp 14.446.890.773.790.020.020.050.07
#4 Exp 22.907.000.010.620.030.010.090.00
#4 Exp 32.142.040.010.010.010.000.010.06
#4 Exp 42.284.440.040.050.030.020.000.00
#5 Exp 10.330.770.020.320.040.050.030.02
#5 Exp 24.375.160.080.720.110.280.111.23
#6 Exp 11.052.631.382.420.000.040.010.00
#6 Exp 22.511.670.024.850.040.060.050.09

Possibly the most striking finding is the ability of actIL15-DC to induce the expansion of MAGE-3- and Tyrosinase-specific CD8+ T cells from naive CD8+ T cells of healthy volunteers. Stimulation with actIL4-DC yielded 0.11 and 0.20% MAGE-3-specific CD8+ T cells in 2/7 healthy volunteers. ActIL15-DC were able to expand the MAGE-3-specific CD8+ T cells up to 0.30 and 0.70% in these two donors, respectively, and expand them up to 0.13 and 1.01% in two further donors (Table 2). Furthermore, actIL15-DC expanded Tyrosinase-specific CD8+ T cells (range 0.3–2.5%) in three volunteers (4/13 experiments), while actIL4-DC expanded Tyrosinase-specific CD8+ T cells up to 0.1% in 1/13 experiments (Fig. 6b, Table 2). A kinetic analysis demonstrated that the expansion of melanoma-specific T cells occurred between days 7 and 12 of culture (Fig. 6c). The superiority of actIL15-DC was not due to mere nonspecific expansion of all T cells because there were no significant differences in the overall recovery of T cells at the end of the cultures ((1.2 ± 0.12) × 106; range 0.84 × 106–2.1 × 106 with IL15-DC vs. (1.0 ± 0.14) × 106; range 0.4 × 106–2 × 106 with IL4-DC; p = 0.1; Fig. 6d).

The superiority of actIL15-DC was also not due to a difference in the ability of the two DC types to load the exogenous peptide, as the actIL15-DC always induced either comparable or higher frequencies of MART-1-specific T cells than IL4-DC, regardless of the amount of peptide (10, 1 or 0.1 µg/mL MART-1 peptide; Fig. 7a, b). ActIL15-DC were significantly more efficient than actIL4-DC at the low MART-1 peptide concentration, i.e. 1 µg/mL rather than 10 µg/mL (Fig. 7b).

Figure 7.

actIL15-DC are superior to actIL4-DC in induction of MART-1-specific CD8+ T cells at low peptide concentration. DC were pulsed with graded concentrations of MART-1 peptide: 10, 1, 0.1 and 0.01 µg/mL. Autologous naive CD8+ T cells were primed as described in Fig. 3. (a) Tetramer staining at day 12 revealed that actIL15-DC were able to expand significant amounts of MART-1-specific CD8+ T cells at peptide concentrations as low as 0.1 µg/mL. (b) Paired observations of five experiments with cells from four healthy volunteers, paired t-test. Significantly higher expansion of MART-1-specific T cells in cultures stimulated with actIL15-DC pulsed with 1 µg/mL of peptide in comparison to actIL4-DC.

Importantly, antigen-specific T cells were functional as they were able to secrete IFN-γ. Supernatants harvested 36 h after the restimulation showed significantly higher amounts of IFN-γ in cultures stimulated by actIL15-DC (Fig. 8, Table 3). In experiments where the MART-1 peptide was titrated, the levels of IFN-γ in the supernatants paralleled the frequency of MART-1-specific T cells, measured by tetramer binding, presented in Fig. 7a/b. At a high peptide dose, the secretion of IFN-γ was comparable; however, at 1 µg/mL of MART-1 peptide, actIL15-DC were more efficient at inducing CD8+ T cells to secrete IFN-γ (Fig. 8, Table 3). Thus, actIL15-DC are particularly efficient at priming functional melanoma-specific CD8+ T cells from healthy volunteers.

Figure 8.

CD8+ T cells primed with IL15-DC make more IFN-γ. Supernatants from DC/T cell co-cultures were harvested 36 h after restimulation on day 7 and analyzed by Luminex. IFN-γ secretion in co-cultures with DC pulsed with a mix of four melanoma peptides (black bars); DC pulsed with 10 µg/mL (striped bar), 1 µg/mL (grey bar), 0.1 µg/mL of MART-1 (white bar), and unpulsed DC (dotted bar). The experiment shown is identical with the experiment depicted in Fig. 6a. Indeed, the higher frequencies of MART-1-specific CD8+ T cells are paralleled by higher amounts of IFN-γ found in the supernatants upon restimulation.

Table 3. CD8+ T cells primed by IL15-DC secrete high amounts of IFN-γ
AntigenIFN-γ (pg/mL)a)aIL4-DC

aIL15-DC

p = 0.04b)

  1. a)IFN-γ was measured by Luminex in supernatants of DC/T cell co-cultures 36 h after restimulation.

  2. b) Paired, two sided t-test.

Four melanoma peptides 10 µg/mLExp 19571818
Exp 218687082
Exp 3327876
MART-1 peptide 10 µg/mLExp 1280503
Exp 2566303
Exp 313744300
MART-1 peptide 1 µg/mLExp 131579
Exp 229150
Exp 32803123
MART-1 peptide 0.1 µg/mLExp 126176
Exp 2037
Exp 3842

IL15-DC turn on potent melanoma antigen-specific CTL

The cytolytic function of cultured HLA-A*0201+ CD8+ T cells was assessed using allogeneic cancer cell lines. In these experiments an equal number of T cells recovered from cultures with IL15-DC or with IL4-DC was tested in a side-by-side comparison. CD8+ T cells, cultured with both activated DC subsets loaded with the four melanoma peptides, efficiently killed the HLA-A*0201+ melanoma cell line Me275 (Fig. 9a). The HLA-A*0201-negative melanoma cell lines Sk-Mel-28, the HLA-A*0201+ breast cancer cell line MCF-7 and the NK cell-sensitive cell line K562 were not killed. T cells expanded with unloaded DC subsets were not able to kill melanoma targets. Incubation of the cell lines with the MHC class I blocking monoclonal antibody W6/32 led to a 45–60% decrease in maximum killing (not shown), thus further confirming CTL-mediated lysis. CTL primed with either actIL15-DC or actIL4-DC were able to kill Me275 melanoma cells (Fig. 9). However, CTL primed in cultures with actIL15-DC appeared more efficient (Fig. 9a; n = 6, p<0.04).

Figure 9.

IL15-DC prime powerful melanoma antigen-specific CTL. Autologous naive CD8+ T cells were cultured, at a 1 : 10 ratio, with DC pulsed with a mix of four melanoma peptides at 10 µg/mL. IL-7 was added at the onset of culture, IL-2 upon restimulation with the relevant DC subsets. (a) CTL activity tested on day 5 after restimulation in a standard 4-h Cr51-release assay using as targets melanoma cell lines: HLA-A*0201+ Me275 and HLA-A*0201 Sk-Mel-28; and control HLA-A*0201+ MCF-7 breast cancer cells and NK-sensitive K562 cells. Cultures with unpulsed DC did not yield CTL (not shown). Paired observations of six independent experiments depicting the maximum killing of each experiment are shown in the final plot (paired t-test). (b) CTL induced by actIL4-DC cannot kill HLA-A*0201+ Sk-Mel-24 melanoma cells (left). CTL induced by actIL15-DC reproducibly killed this cell line (middle and final), but not K562 or the HLA-A*0201 Colo829 melanoma cells (paired t-test; p <0.03).

The melanoma cell line Sk-Mel-24 is a HLA-A*0201+ cell line that, in our hands, has proven until now to be resistant to all the CTL we have been able to generate, independent of the type of antigen-presenting cell used. Accordingly, actIL4-DC-stimulated CTL did not lead to a specific killing (Fig. 9b). However, CTL generated from cultures with four melanoma peptide-loaded actIL15-DC reproducibly killed this cell line efficiently (Fig. 9b; n = 4, p <0.03). Thus, actIL15-DC permit the generation of highly efficient melanoma-specific CTL.

IL15-DC express cytokines that might help CD8+ T cell differentiation

CD8+ T cell differentiation was depending upon cell-cell contact, suggesting a role of membrane-bound molecules. Therefore, highly purified (>98%) IL4- and IL15-DC, non-activated and LPS-activated, were sonicated and the resulting supernatants were analyzed using multiplex cytokine analysis. Of the 23 cytokines/chemokines that were analyzed, we focus on those that could be relevant to T cell differentiation. As shown in Fig. 10, IL4-DC and IL15-DC reciprocally expressed IL-4 and IL-15 (Fig. 10), consistent with the cytokines used to generate these myeloid DC. We did not detect differences in the expression of IL-10 or IL-12p70 in actIL4-DC and actIL15-DC (not shown). Similarly, the two T cell-related cytokines IFN-γ and IL-2 could be detected in both IL4- and IL15-DC (not shown). In three of four cultures generated from different healthy volunteers, actIL15-DC uniquely expressed high amounts of IL-1α, IL-1β and IL-8. Detection of IL-15 in sonicated cells (Fig. 10) but not in the culture supernatants (not shown) suggests the presence of membrane-bound IL-15. This form of IL-15 could be biologically active, as described previously 1517, and therefore contribute to enhanced CD8+ T cell priming and differentiation.

Figure 10.

IL15-DC express cytokines that might help CTL priming. IL4-DC and IL15-DC were generated from purified monocytes and further sorted after culture to eliminate the containing lymphocytes (>98% purity). Sorted cells were sonicated and the obtained supernatants were analyzed with multiplex cytokine assay analyzing 23 cytokines. Expression of selected cytokines (ordinate, pg/mL) is shown for each of the tested conditions (abscissa) for each of the four independent experiments using cells generated from four different donors (color code). Paired t-test.

Discussion

Our study demonstrates that IL15-DC are more efficient than IL4-DC in generation of melanoma-specific CTL, as measured by several parameters of CD8+ T cell immunity including: (i) priming of an extended repertoire of melanoma antigen-specific CD8+ T cells, (ii) enhanced expression of type I cytokines (IFN-γ) and reduced expression of type II cytokines (IL-4) by primed T cells, (iii) enhanced expression of Granzyme B and Perforin by primed T cells, and (iv) enhanced capacity of primed T cells to kill “CTL-resistant” melanoma cells.

Both activated IL-4- and IL-15-induced DC can expand high-frequency antigen-specific naive CD8+ T cells specific for MART-1. However, actIL15-DC are more efficient than actIL4-DC for the expansion of CD8+ T cells specific for Tyrosinase or MAGE-3. IL15-DC also affect the quality of melanoma-specific T cells and polarize them towards (i) type I immunity with high secretion of IFN-γ and absence of IL-4 and (ii) high expression of Granzyme B and Perforin indicating enhanced CTL maturation. Accordingly, these CTL can kill Sk-Mel-24 melanoma cells, which we find consistently resistant to lysis by CD8+ T cells primed and/or restimulated with antigen-loaded actIL4-DC.

IL-15 itself induces in vitro antigen-independent expansion of human naive CD8+ T cells 18 and enhances antigen-specific proliferation of T cells isolated from HIV-infected patients 19. Yet, the mere action of soluble IL-15 cannot explain the enhanced priming by IL15-DC, as the preferential expansion of CD8+ T cells depends upon cell-cell contact. Flow cytometry analysis indicated that the enhanced generation of CTL cannot be simply explained by the enhanced expression of MHC class I and/or costimulatory molecules by IL15-DC. A more mature stage of IL15-DC cannot be considered either, as LPS-activated IL4-DC express higher levels of CD83, a classical marker of DC maturation. Thus, IL-15 might induce a unique set of DC molecules that facilitate priming. In addition, IL-15 itself acts, in the membrane-bound form, as a costimulatory molecule 1517. Indeed, sonicated IL15-DC express IL-15, suggesting the presence of a membrane-bound form that might be biologically active and therefore contribute to enhanced CTL priming. Two recent studies demonstrate that IL-15 expressed by human DC is important in the expansion of effector cells, either virus-specific noncytotoxic CD8+ T cells in HIV or NK cells in secondary lymphoid organs 20, 21. Interestingly, distinct aspects of NK cell biology appeared differentially regulated by DC-derived cytokines. Thus, NK cell proliferation was dependent upon membrane-bound IL-15 while NK cell differentiation to IFN-γ-secreting cells was mediated by IL-12 21. It remains to be determined whether distinct aspects of CD8+ T cell expansion and differentiation would be similarly regulated.

actIL15-DC uniquely express high amounts of IL-8, which might further contribute to their capacity to expand antigen-specific CTL. Indeed, recent studies have identified a subset of human CD8+ T cells that express CXCR1, respond to IL-8 and have cytotoxic function 22, 23. Memory CD8+ T cells do not express this receptor 22, 23. Thus, IL15-DC might preferentially attract this subset of CD8+ T cells, particularly upon secondary stimulation.

The demonstration that IL15-DC prime CTL that can overcome escape mechanisms and kill otherwise resistant tumors provides an important parameter for pre-clinical assessment of DC vaccines. Cancer cells, including melanomas, have evolved to escape CTL lysis (reviewed in 24). Beside the down-regulation of MHC class I expression and/or selection of tumor antigen-negative variants 24, tumor cells overexpress serine protease inhibitors that block the Granzyme B/Perforin pathway 25, or the anti-apoptotic protein c-FLIP that protects cells from apoptosis induced via death receptors 26. Thus, a further understanding of the molecular mechanisms by which IL15-DC-primed T cells overcome tumor resistance may eventually lead us to establish the parameters of “optimal” CTL. Candidates under consideration include PD-1/PD-1 ligand (B7-H1) interaction, as exogenous IL-15 has been shown to rescue CD4+ T cells from inhibition mediated by PD-1 engagement 27. Another explanation might come from the balance of inhibitory and activating NK cell receptors expressed on CTL 28 that may be very different on T cells primed by these two distinct DC subsets.

Our results show that IL15-DC are efficient in the generation of tumor-specific effectors. It will be important to determine whether they can prime long-lasting tumor-specific memory T cells, which might be important for the control of tumor relapse. In this context, co-administration of IL-15, but not IL-2, with HIV vaccines delivered in vaccinia vectors led to priming of long-lived antigen-specific memory CD8+ T cells 29. We and others have recently shown in mice the potency of IL-15-induced DC to generate OVA-specific immunity in vivo3032. Furthermore, we showed that human IL15-DC were particularly efficient at activating Flu matrix peptide-specific memory CD8+ T cells 14. The importance of the current work stems from the demonstration of efficient priming of CTL specific to self antigens expressed by tumor cells. This makes IL15-DC interesting candidates for DC-based vaccination protocols.

Materials and methods

DC generation

Monocytes were enriched by adherence or purified by elutriation (for cytokine analysis). Enriched monocytes were cultured in complete RPMI 1640 (GIBCO BRL) supplemented with 5–10% autologous serum, GM-CSF (Immunex; 100 ng/mL, U/mL) and either IL-4 or IL-15 (R&D Systems) at 25 ng/mL (U/mL) and 200 ng/mL (U/mL), respectively. DC were activated by adding LPS (Sigma) at 10 ng/mL. Immature DC were used at day 3 and activated DC at day 4. Phenotype analysis: HLA-DR, CD11c, CD14, CD209, CD80, CD56, CD16, CD8, CD3, CD4 (BDIS); CD207, CD3, CD40 (Beckman-Coulter); CD86, CD83 (Pharmingen); HLA-ABC (Dako); CCR6, CCR7 (R&D Systems). For assessment of cytokine secretion generated DC were sorted to insure purity (>98%) and sonicated to measure membrane-bound and intracellular cytokine expression using multiplex cytokine analysis as described below.

Cell lines

K562 cells, Sk-Mel-24, Sk-Mel-28 and Colo829 melanoma cell lines, the MCF-7 breast cancer cell line and T2 cells were from the American Type Culture Collection (ATCC). The Me275 and Me290 melanoma cell lines were a kind gift of Drs. J.-C. Cerottini and D. Rimoldi. All cell lines were maintained in cRPMI + 10% fetal calf serum.

DC/lymphocyte co-cultures

Allogeneic PBL were obtained by depleting PBMC of CD14+, BDCA4+ and Glycophorin A+ cells using microbeads (Miltenyi Biotech). CD8+ T cells were purified (>95%) by positive selection after depletion of CD56+/CD16+ cells. Autologous naive CCR7+ CD45RA+ CD56 CD16 CD8+ T cells (>95% purity) (IRB 097-053) were sorted from PBMC depleted of CD4+, CD56+, CD16+, CD19+, CD14+ cells.

In experiments with immature DC, allogeneic lymphocytes were primed and restimulated once. In experiments with activated DC, allogeneic lymphocytes were primed without exogenous cytokines and assessed at day 7. Priming of autologous T cells was done at a 10 : 1 ratio with IL-7 (10 IU/mL) from the onset of culture and IL-2 (10 IU/mL) after restimulation on day 7. Contamination of CD8+ T cell cultures by NK cells and CD4+ T cells was <1.2 and 2%, respectively, in IL15-DC cultures.

T cell assessment

Streptavidin-PE or APC-labeled tetramers were from Beckman Coulter. T cells (1.5 × 105 to 2.5 × 105) were stained with: CD45RO-FITC, tetramer-PE, CD3-PerCP, CD8-APC at room temperature in the dark for 25 min. HIV(gag)-specific tetramer and the identical staining in unloaded-DC conditions served as controls (not shown). Intra-experimental standard deviation of triplicate wells of the same DC subset condition ranged between 0.4 and 1.7% for MART-1-positive T cells, between 0.04 and 0.08% for gp100- and <0.08% for MAGE-3- and Tyrosinase-specific CD8+ T cells. For intracellular staining, the specific tetramer was labeled with APC, CD8-PerCP and the cytokines/effector molecules were stained with specific antibodies after permeabilization using the BD Cytofix/Cytoperm kit according to the manufacturer's recommendations.

For CFSE labeling, cells (1 × 106–5 × 106/mL) were stained, upon restimulation of cultures with DC or at the onset of culture, at room temperature in the dark at a final concentration of 2.5 µM of CFSE for 10 min. Cytotoxicity was measured with a standard 4-h chromium51 (Cr51)-release assay. Targets were labeled for 1 h with Cr51 (NEN Life Science Products) and incubated, at different ratios with effector cells, at 37°C in a total volume of 200 µL of cRPMI/10% AB serum. The percentage of specific lysis was calculated as (cpmexperiment − cpmspontaneous release) / (cpmmaximum release − cpmspontaneous release). Anti-HLA ABC mAb (clone W6/32; DAKO) at 50 µg/mL or isotype control purified mouse IgG2a (Becton Dickinson) at 50 µg/mL were added at the onset of the cytotoxicity assay. The peptides used to pulse DC were: MART-1 AAGIGILTV27–35 (10, 1, 0.1, 0.01 µg/mL), gp100 IMDQVPFSVg209–2M (10 µg/mL), MAGE-3 FLWGPRALV271–279 (10 µg/mL), Tyrosinase YMDGTMSQV368–376 (10 µg/mL). Cells from seven consecutive HLA-A*0201-positive healthy volunteers were tested in altogether 13 experiments to determine reproducibility (Table 3).

Cytokine multiplex analysis

Cytokine multiplex analysis was carried out using the Beadlyte cytokine assay kit (Upstate) as per the manufacturer's protocol. Cytokine concentrations were measured with a Bio-Plex Luminex 100 XYP instrument and calculated using Bio-Plex Manager 3.0 software with a 5-parameter curve-fitting algorithm applied for standard curve calculations.

Statistical analysis

Two-sided, paired t-test and Wilcoxon matched pairs test were used.

Acknowledgements

We thank Dr. Joseph Fay, Bi-Jue Chang, Nathalie Piqueras, Doris Wood, and Susan Hicks for help with recruitment and follow up of healthy volunteers. We thank Lynnette Walters at BIIR Cell and Tissue Procurement Core; Susan Burkeholder, Jennifer Finholt-Perry and Fabienne Kerneis at BIIR GMP Lab; and Elizabeth T. Kraus and Sebastien Coquery at BIIR Flow Cytometry Core for technical help; Cindy Samuelsen and Nicolas Taquet for invaluable help. We thank Dr. Michael Ramsay for continuous support. Supported by Baylor Health Care Systems Foundation, NIH (U19 AIO57234, CA78846 and CA085540: J.B., PO-1 CA84512: J.B./A.K.P., CA89440: A.K.P.), FWFAustria (Schroedinger Stipendium, J2255-B08: P.D.). J.B. holds the Caruth Chair for Transplant Immunology Research. A.K.P. holds the Ramsay Chair for Cancer Immunology Research.

Footnotes

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