A Soluble Form of Lymphocyte Activation Gene-3 (IMP321) Induces Activation of a Large Range of Human Effector Cytotoxic Cells

Blood collection and PBMC isolation

Blood samples from healthy volunteers and metastatic cancer patients (6 breast and 15 renal carcinomas) with informed consent were collected in heparinated lithium tubes (Vacutainer; BD Biosciences) or were obtained from buffy coats provided by EFS. PBMCs were isolated by Ficoll-Paque density gradient (Pharmacia) using LeucoSep tubes (Greiner Bio-One) and cryopreserved in FCS (HyClone) containing 10% DMSO (Sigma-Aldrich) or were used immediately.

Reagents

Recombinant soluble human LAG-3Ig fusion protein (IMP321), a 200-kDa dimer, was produced in Chinese hamster ovary cells transfected with a plasmid encoding for the extracellular domain of human LAG-3 fused to the human IgG1 Fc. The purified bulk preclinical batch has a concentration of 1.9 mg/ml IMP321 and 0.75 EU/mg of endotoxin, 0.42 ng/ml DNA, and 6 ng/ml host cell proteins. Human IgG1 (hIgG1) was obtained from Chemicon International. IMP321 and hIgG1 were conjugated to Alexa Fluor 488 using the FluoReporter protein labeling kit from Molecular Probes. The series of TLR agonists was purchased from Apotech. The anti-LAG-3 mAb (clone 17B4, IgG1) directed to the extra loop of the Ig-like domain 1 of LAG-3 (18) has been characterized previously (19, 20). This mAb was FITC labeled using FluoReporter FITC protein labeling kit from Molecular Probes. A second anti-LAG-3 mAb (clone A9H12, IgG2a) was obtained by immunization of mice with CHO cells transfected with LAG-3 and boosted with sLAG-3Ig. Isotype-matched control mAbs (mIgG2a and mIgG1) were purchased from Southern Biotechnology Associates. The M-DC8 mAb (IgM) has been characterized previously (21, 22). FITC-conjugated anti-IFN-γ (25723.11), TNF-α (6401.1111), granzyme A (CB9) and granzyme B (GB11), PE-conjugated CD1a (SK9), CD11b (D12), CD11c (B-ly6), CD13 (Leu-M7), CD14 (MφP9), CD16 (Leu 11c), CD33 (P67.6), CD45RA (HI100), CD56 (MY31), CD123 (9F5), anti-HLA-DR (L243), TCRγ/TCRδ (11F2), MCP-1 (5D3-F7), CCL4/MIP-1β (D21-1351), IL-2 (5344.11), and IFN-γ (25723.11), PerCP-Cy5.5-conjugated CD3 (SK7) and CD19 (J25C1), PerCP-conjugated CD14 (MφP9), PE-Cy7-conjugated CD4 (SK3), CD16 (B73.1), CD45RA (L48), and CD56 (B159), allophycocyanin-conjugated anti-TNF-α (6401.1111), CD11a (HI111), CD11c (S-HCL-3), CD14 (MφP9), and CD27 (L128), Alexa 647-conjugated anti-CCR7 (3D12) and allophycocyanin-Cy7-conjugated CD8 (SK1) and anti-HLA-DR (L243) mAbs, brefeldin A, CytoFix/CytoPerm, and Perm/Wash buffers were purchased from BD Biosciences. PE-conjugated CD4 (13B8.2), CD80 (MAB104), CD83 (HB15a), and CD86 (HA5.2B7) were obtained from Coulter Immunotech and PE-conjugated anti-HLA-DP/DQ/DR (WR18) mAb was obtained from Abcam.

Binding assay of IMP321

PMBCs were incubated with MDC-8 mAb, washed, and labeled with anti-IgM-PE (Immunotech). Then cells were incubated in PBS containing 10% human AB serum (Bio Media), 0.1% azide for 30 min at 4°C to block unspecific binding sites, and then with Alexa 488-conjugated IMP321 or hIgG1 at a final concentration of 3 μg/ml in PBS, 0.5% BSA, 0.1% azide containing 30 μg/ml unlabeled hIgG1 and simultaneously stained with CD14-PerCP, CD11c-allophycocyanin, CD16-PE-Cy7 and anti-HLA-DR-allophycocyanin-Cy7 mAbs for 30 min at 4°C. Cells were washed in PBS BSA and analyzed using a FACSCanto flow cytometer (BD Biosciences). In inhibition assays, 3 μg/ml Alexa 488-conjugated IMP321 or hIgG1 were incubated with anti-LAG-3 mAb (A9H12) or control mIgG2a at 30 μg/ml for 30 min at room temperature before adding the cells.

Cytometric bead array (CBA) assay

PBMCs were incubated in complete RPMI 1640 containing 10% FCS with or without IMP321 (1 μg/ml or indicated concentration), hIgG1 (1 μg/ml), anti-MHC class II mAb (1 μg/ml, clone I3; Coulter), or TLR agonists (concentrations are indicated as optimal by the provider): TLR1-2 (Pam₃Cys-Ser-(Lys)₄, 0.1 μg/ml), TLR3 (polyinosinic-polycytidylic acid, 100 μg/ml), TLR4 (LPS, 1 μg/ml), TLR5 (flagellin, 0.1 μg/ml), TLR6-2 (macrophage-activating lipopeptide-2, 0.1 μg/ml), TLR7-8 (polyuridylic acid, 10 μg/ml), or TLR9 (CpG oligodeoxynucleotide 2395, 10 μg/ml) for 24 or 48 h as indicated. IFN-γ, TNF-α, IL-1β, IL-6, CCL4, CCL5, CCL2, and IL-10 were quantified in culture supernatants by specific CBA Flex sets according to manufacturer’s instructions (BD Biosciences). Data were acquired with FACSCanto cytometer and cytokines/chemokines concentrations were calculated using FCAP software (BD Biosciences). Results are reported as a quantity of analytes produced by 10⁶ cells.

PBMC stimulation and intracellular staining

PBMCs were stimulated with IMP321 (1 μg/ml or indicated concentration), hIgG1 (1 μg/ml), anti-MHC II mAb (1 μg/ml, clone L243, or clone I3; BD Biosciences) or TLR agonists (see previous section) in complete RPMI 1640 10% FCS for 18 h in the presence of brefeldin A during the last 16 h or for 4 h in the presence of brefeldin A (to evaluate myeloid cells activation) in a humidified incubator at 37°C. In neutralization assays, indicated concentrations of IMP321 were incubated with anti-LAG-3 mAb (17B4, 10 μg/ml) or control mIgG1 (10 μg/ml) for 30 min at room temperature before adding the cells. After stimulation, cells were washed and labeled with fluorochrome-conjugated specific Abs for CD4, CD8, CD11a, CD11c, CD14, CD16, CD27, CD28, CD45RA, HLA-DR, and/or CCR7 as indicated in the figure legends. Cells were fixed and permeabilized using CytoFix/Cytoperm, washed with Perm/Wash buffer, and then stained for cytokines/chemokines, and CD3 when required. After extensive washing with Perm/Wash buffer, cells were analyzed using a six-color FACSCanto flow cytometer to determine the percentage of cells expressing cytokines/chemokines and to phenotype the corresponding activated cells. The results following FACS analysis are defined as the differences between the percentage of cells expressing at least one cytokine upon IMP321 stimulation and the percentage of cytokine⁺ cells in unstimulated conditions. The confidence interval depended on the numbers of relevant events (CD3⁺CD8⁺ or CD3⁻CD8⁺ events) collected in each sample, the background expression of cytokines in unstimulated PBMC and the difference between the two conditions. This difference was significant with a power of 90% (p < 0.05) if the number of CD3⁺CD8⁺ or CD3⁻CD8⁺ cells collected by the cytometer in a given sample was larger than calculated events using the formula: where Stim is the percentage of CD3⁺CD8⁺ or CD3⁻CD8⁺ cells expressing at least one cytokine upon stimulation with IMP321 and Unstim is the percentage of CD3⁺CD8⁺ or CD3⁻CD8⁺ cells expressing at least one cytokine in basal condition.

Isolation of CD8⁺ T cell populations from PBMCs

PBMCs were thawed and stained with PE-conjugated CD4, CD14, CD16, CD56, anti-TCRγ/anti-TCRδ, HLA-DR and HLA-DR/DQ/DP, CD3-PerCP-Cy5.5 and CD8-allophycocyanin-Cy7, washed, and CD3⁺CD8⁺ cells were sorted after exclusion of debris, doublets, and PE-positive cells using a FACSAria cell sorter (BD Biosciences). Resulting preparations were consistently 96% CD3⁺CD8⁺ and the remaining events consisted of debris. Whole PBMCs and CD3⁺CD8⁺ sorted cells were then incubated with or without IMP321 for 18 h with brefeldin A for the last 16 h, stained for CD3, CD4, CD8, IFN-γ, and TNF-α and analyzed by cytometry as described above.

Proliferation assay

Fresh PBMCs were washed in PBS, resuspended at 2 × 10⁶/ml in PBS, stained with CFSE (50 nM) for 10 min at 37°C in the dark and washed in RPMI 1640 containing 10% FCS (Invitrogen Life Technologies). Cells were incubated with or without IMP321 (10 μg/ml) at 37 °C. After 4 days, T cell subpopulations were stained with CD4-PE-Cy7 and CD8-allophycocyanin-Cy7 Abs and analyzed by flow cytometry. The percentage of CD4⁺ and CD8⁺ CFSE^low cells corresponding to dividing cells was determined using the ModFit software (Verity Software House). Weighted scores are calculated by multiplying the total percentage of dividing CFSE^low cells by the number of cell divisions.

Binding of IMP321 to PBMCs

Alexa 488-labeled IMP321 bound to a minority (<10%) of MHC class II⁺ cells, representing 0.5–5.6% of total PBMCs in seven experiments (Fig. 1⇓A, upper panel). IMP321⁻ and IMP321⁺ cells were then phenotyped separately. In the IMP321⁺HLA-DR⁺CD11c⁺ population, the percentages of CD16⁺CD14⁻ or CD16⁺CD14^int were variable from one subject to another but these remained the main subsets (Fig. 1⇓A, right panels). This IMP321⁺HLA-DR⁺CD11c⁺CD16⁺ subset also expressed CD4, CD13, CD33, CD45RA, and CD86 but not CD1a, CD3, CD19, CD80, CD83, or CD123 (data not shown). Thus, the main IMP321⁺ cell subset includes cells phenotypically defined as myeloid CD16⁺ DCs (23). Of note, all myeloid CD11c⁺CD16⁺ DCs in PBMCs as well as almost all M-DC8⁺ cells, the major DC subpopulation in blood (21, 22), were IMP321⁺. In addition, a small percentage of IMP321⁺ cells were HLA-DR⁺CD11c⁺CD16⁻CD14^high monocytes (Fig. 1⇓, right panel) and accordingly most monocytes were IMP321⁻ cells even though they express MHC class II molecules. The same happens for B lymphocytes with only a small percentage (<5%) of CD19⁺ cells being IMP321⁺ (data not shown). Preincubation of IMP321 with a LAG-3 Ab inhibited its binding to PBMCs (Fig. 1⇓B) showing that the LAG-3 domain and not the Fc portion was responsible for its binding to APCs. Also, the three CD16 dot-plot histograms in Fig. 1⇓ clearly show that cells lacking this FcR do bind IMP321.

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FIGURE 1.

Binding of IMP321 to PBMCs. PBMCs were stained with M-DC8/anti-IgM-PE, then incubated with Alexa 488-hIgG1 (dashed line) or Alexa 488-IMP321 (filled histogram) (3 μg/ml), stained with CD14-PerCP, CD11c-allophycocyanin, CD16-PE-Cy7, and HLA-DR-allophycocyanin-Cy7 mAbs and analyzed on a six-color cytometer. After exclusion of doublets, debris, and dead cells based on forward and side scatter parameters, IMP321⁻ (shown in gray) and IMP321⁺ (black) cells were gated and analyzed separately (A, left). Dot plots showing the expression of HLA-DR/CD11c, CD14/CD16, CD11c/CD16, HLA-DR/CD16, and IMP321/M-DC8 of IMP321⁻ cells (gray) and IMP321⁺ cells (black) are presented in the right panels. Data are representative of at least four donors. Inhibition of Alexa 488-IMP321 binding to PBMCs was done by preincubation of Alexa 488-IMP321 with an anti-LAG-3 (black line) vs a control Ab (filled histogram). Then the mixture was used to stain the cells as described above (B).

IMP321 induces PBMCs to produce cytokines/chemokines, but not IL-10

The individual quantitative analysis (CBA assay) of a series of 48-h PBMC supernatants showed that IMP321 at 1 μg/ml consistently induced a 1–3 orders-of-magnitude increase in the concentrations of IFN-γ, TNF-α, IL-1β, IL-6, CCL4, CCL2, and CCL5 (Fig. 2⇓A). Of note, IL-10 was not induced and in some donors even decreased compared with the unstimulated condition (Fig. 2⇓A). In dose-response experiments, the increase in CCL4 at 24 h was detected with IMP321 concentrations as low as 10 ng/ml (Fig. 2⇓B) whereas stimulation of PBMC with 1 μg/ml hIgG1 had no effect (n = 8, data not shown). In the same conditions, cross-linking MHC class II molecules with a specific mAb (clone I3) at 1 μg/ml induced only a slight increase (<10% of the IMP321 effect) of IFN-γ, TNF-α, IL-1β, CCL2, and CCL5 (n = 4, data not shown).

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FIGURE 2.

IMP321 induces PBMCs to produce cytokines/chemokines. Quantitative measurement of IFN-γ, TNF-α, IL-1β, IL-6, IL-10, CCL2, CCL4, and CCL5 in 48-h supernatants of unstimulated (medium) and IMP321-stimulated (1 μg/ml) PBMCs from 24 donors (9 for CCL2) were determined by CBA assays. Data represent the concentrations of cytokine/chemokine for each donor (circles) and the medians obtained for all donors (bars), expressed in picograms per 10⁶ cells on a logarithmic scale (A). Eight PBMC samples were stimulated with the indicated concentrations of IMP321 for 24 h and the concentration of CCL4 in the supernatants was determined by CBA. Data represent the mean ± SD of CCL4 concentrations determined for all PBMC samples, expressed in picograms per 10⁶ cells on a logarithmic scale and plotted as a function of IMP321 concentration (B).

IMP321 induces myeloid cells to produce cytokines/chemokines

The effect of IMP321 on cytokine/chemokine production in different cell subsets was assessed by intracellular staining followed by flow cytometry analysis. PBMCs were stimulated with IMP321 for 4 h, stained with HLA-DR and CD11c mAb, and permeabilized to detect the intracellular expression of TNF-α and CCL4. IMP321, but not the control hIgG1, induced the expression of CCL4 and/or TNF-α in ∼5% of PBMCs (Fig. 3⇓, upper panel). Cells expressing both CCL4 and TNF-α were all HLA-DR⁺CD11c⁺ myeloid cells (Fig. 3⇓, lower panel).

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FIGURE 3.

IMP321 induces myeloid cells to produce cytokines/chemokines. PBMCs incubated with hIgG1 or IMP321 (1 μg/ml) for 4 h in the presence of brefeldin A were stained with anti-HLA-DR-allophycocyanin-Cy7 and CD11c-allophycocyanin mAbs, fixed, permeabilized, and then stained with anti-TNF-α-FITC and CCL4-PE mAbs. The percentages of PBMCs expressing CCL4 and/or TNF-α and the phenotype of these cells were determined by flow cytometry. Dot plots represent the expression of TNF-α and CCL4 on PBMCs gated on forward and side scatter parameters after exclusion of doublets and debris (upper panels). Cells expressing CCL4 and TNF-α are shown in black, cells expressing one of these cytokines are shown in dark gray, and cells expressing none of these cytokines are shown in gray. Lower panel, The expression of HLA-DR/CD11c on the CCL4⁺TNF-α⁺ cells (black), the single cytokine⁺ cells (dark gray) and the cytokines⁻ cells (gray). Results are representative of six donors.

IMP321 induces CD8⁺ T cells and NK cells to produce cytokines/chemokines

After PBMC stimulation (60 donors) with IMP321 for 18 h, cells were stained with CD3, CD4, CD8 mAb and with IFN-γ, TNF-α, and IL-2 mAb after permeabilization of the cell membrane. The percentages of CD3⁺CD4⁺, CD3⁺CD8⁺ T cells, and CD3⁻CD8⁺ NK cells expressing one, two, or three of these cytokines were determined. The measurement of cytokine⁺ cells using this procedure has been validated previously and gives an overall coefficient of variation <20% after Ag-specific stimulation (24). A slight increase in cytokine-expressing CD4⁺ T cells was detected in 44 (of 60) PBMCs stimulated with IMP321 compared with medium alone. Of these, 14 displayed a significant increase (data not shown). In contrast, a much higher percentage of cytokine-expressing CD8⁺ T cells was observed (Fig. 4⇓A, upper panel). Indeed, IMP321 induced a significant percentage of IFN-γ⁺ or IFN-γ⁺TNF-α⁺ activated CD8 T cells in most donors (46 of 60). We have compared this to the response of CD8 T cells to a pool of CMV peptides in 24 of these 60 donors and found that the range of values shown in Fig. 4⇓A is quite similar to the one observed in response to CMV stimulation (data not shown). The effect of IMP321 was also determined on CD3⁻CD8⁺ cells, a subset of NK cells (Fig. 4⇓A, lower panel). A significant increase in the percentage of such cells expressing IFN-γ and/or TNF-α was detected in 52 of 60 donors.

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FIGURE 4.

Induction of activated CD8⁺ T and NK cells by IMP321 in PBMCs from 60 healthy donors and 21 metastatic cancer patients. PBMCs incubated with IMP321 (1 μg/ml) for 18 h were stained with CD4-PE-Cy7, CD8-allophycocyanin-Cy7, fixed, permeabilized and stained with CD3-PerCP-Cy5.5, anti-IFN-γ-FITC, IL-2-PE, and TNF-α-allophycocyanin mAbs. The percentage of CD3⁺CD8⁺ T lymphocytes and CD3⁻CD8⁺ NK cells expressing IFN-γ, TNF-α, and/or IL-2 was determined by flow cytometry. Percentage of cells expressing cytokines in unstimulated conditions was subtracted from IMP321-stimulated percentage. Because no significant IL-2 production was induced in CD3⁺CD8⁺ or CD3⁻CD8⁺, these data are not presented. The percentages of IFN-γ⁺TNF-α⁻, IFN-γ⁻TNF-α⁺, and IFN-γ⁺TNF-α⁺ in CD3⁺CD8⁺ and CD3⁻CD8⁺ subpopulations are presented for every subject (A). An example of IFN-γ and TNF-α expression in CD3⁺CD8⁺ (B, upper left) and CD3⁻CD8⁺ cells (B, upper right) in one donor and the mean ± SEM of the percentage of cells expressing IFN-γ and/or TNF-α for the 60 healthy donors are shown (B). In dot plots, the percentages of cells in IMP321-stimulated and unstimulated conditions are indicated in each quadrant in bold and in parentheses, respectively. PBMCs from 21 metastatic cancer patients were stimulated or not with IMP321 (1 μg/ml) and then were stained and analyzed as described in A. Data represent the percentage of CD3⁺CD8⁺ (C, left) and CD3⁻CD8⁺ cells (C, right) expressing at least one of the cytokines (IFN-γ, IL-2, and TNF-α) for healthy donors ≥40 years old and the 21 patients as indicated. Bars represent the mean in each group. ∗, A significant difference between the two groups (Student’s t test, p < 0.05).

IFN-γ and TNF-α expression in CD3⁺CD8⁺ and CD3⁻CD8⁺ cells for one donor is shown as an example in Fig. 4⇑B (upper panels). The average values over 60 donors are 1 ± 0.2% and 3.7 ± 0.4% for CD3⁺CD8⁺ and CD3⁻CD8⁺ cells, respectively, vs 0.2 ± 0.1% and 0.4 ± 0.004% in medium alone (Fig. 4⇑B, lower panels).

This induction of Tc1 cytokines was also investigated in 21 untreated metastatic cancer patients where an immunosuppressive environment might interfere with the observed effect of IMP321. Because the majority of the healthy subjects were much younger than the cancer patients, only the healthy subjects >40 years old (n = 13) were included in the comparison. The percentages of CD8⁺ T cells expressing IFN-γ ⁺ and/or TNF-α⁺ upon IMP321 stimulation were similar (Fig. 4⇑C, left panel) while the percentages of CD8⁺ NK cells were significantly lower in cancer patients compared with healthy donors (Fig. 4⇑C, right panel).

Similar frequencies were observed for CD3⁺CD8⁺TCRγ/CD3⁺CD8⁺TCRδ⁺ T cells (data not shown) or for the whole NK population (CD3⁻CD56⁺ cells; Fig. 5⇓A). IMP321 also induces the production of CCL4 in a large percentage of CD8⁺ T cells (>2%) and NK CD8⁺ cells (>26%) in the three donors analyzed (Fig. 5⇓, B and C). All cells expressing type 1 cytokines are included into the CCL4⁺ subset (Fig. 5⇓, B and C).

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FIGURE 5.

IMP321 induces CD8⁺ T cells and NK cells to produce cytokines/chemokines. PBMCs (from one representative subject of six tested) were stimulated with or without IMP321 (1 μg/ml) and stained with CD56-PE-Cy7, CD8-allophycocyanin-Cy7, fixed, permeabilized, and then stained with CD3-PerCP-Cy5.5, anti-IFN-γ-FITC, and TNF-α-allophycocyanin mAbs to show the expression of these cytokines in the whole NK cells population (CD3⁻CD56⁺), the CD8⁻ NK cells subset (CD3⁻ CD56⁺CD8⁻) vs the CD8⁺ NK cells subset (CD3⁻CD56⁺CD8⁺) (A). IMP321-stimulated PBMCs were stained with CD4-PE-Cy7, CD8-allophycocyanin-Cy7, fixed, permeabilized, and then stained with CD3-PerCP-Cy5.5, anti-IFN-γ-FITC, CCL4-PE, and TNF-α-allophycocyanin mAbs (B and C). Dot plot histograms showing the expression of IFN-γ, TNF-α, and CCL4 in CD3⁺CD8⁺ cells (B) and CD3⁻CD8⁺ cells (C) in a representative subject (of three) are presented. In dot plot histograms, the percentages of cells in IMP321-stimulated and unstimulated conditions are indicated in each quadrant in bold and in parentheses, respectively.

In the intracellular staining assay, neither human IgG1 (n = 8) nor MHC class II mAb (L243 or I3, n = 2) had any effect on expression of intracellular cytokines in CD8⁺ T cells (Fig. 6⇓A). The Tc1 activation of CD8⁺ T cells by IMP321 was detectable at concentrations as low as 30 ng/ml and was inhibited by the prior addition to the PBMCs of a blocking LAG-3 mAb (Fig. 6⇓B). Pure sorted populations of CD3⁺CD8⁺ cells could not be induced by IMP321 to produce IFN-γ or TNF-α (Fig. 6⇓C), underlining the role of MHC class II⁺ APC activation by IMP321 in Tc1 activation.

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FIGURE 6.

Specific and indirect CD8⁺ T cell activation by IMP321. PBMCs were stimulated with hIgG1 (1 μg/ml), anti-MHC class II Abs (I3 or L243, 1 μg/ml), or IMP321 (1 μg/ml) for 18 h and were stained as described in Fig. 4⇑A. The percentages of IFN-γ⁺TNF-α⁻, IFN-γ⁻TNF-α⁺, and IFN-γ⁺TNF-α⁺ in CD3⁺CD8⁺ cells are presented (A). Different concentrations of IMP321 were preincubated with 10 μg/ml LAG-3 mAb (+) or isotype-matched mAb (−) before addition of PBMCs. After overnight stimulation, PBMCs were stained as described in Fig. 4⇑A and the percentages of CD3⁺CD8⁺ cells expressing IFN-γ and TNF-α were plotted as a function of IMP321 concentrations with or without the anti-LAG-3 mAb (B). CD3⁺CD8⁺ T cells were sorted from whole PBMCs after exclusion of debris, doublets, CD4⁺, CD14⁺, CD16⁺, CD56⁺, TCRγ/TCRδ⁺, HLA-DR⁺, and HLA-DR/DP/DQ⁺ cells. Whole PBMCs and CD3⁺CD8⁺ sorted cells were then incubated with or without IMP321 and stained for CD3, CD4, CD8, IFN-γ, and TNF-α (C). CFSE-labeled PBMCs from two donors were incubated with or without IMP321. After 4 days, CD8⁺ cells were stained with specific Abs and the number of cell divisions in CD8⁺ population was analyzed using Modfit software. The percentage of CFSE^low cells is indicated (D).

Effect of IMP321 on T cell proliferation

In addition to these early cell activation events, we also studied the later proliferative effect of IMP321. PBMCs were first labeled with CFSE, incubated with IMP321, and analyzed on day 4 for the number of cell divisions. IMP321 induced more CD8⁺ T cells to divide (10 and 8% compared with 4 and 3%, respectively, for the two donors shown in Fig. 6⇑D). In addition to this, the proliferating cells divide more (up to generation 5) in the presence of IMP321. Weighted scores calculated by multiplying the total percent divisions of CFSE^low by the number of cell divisions were 5 and 3% in unstimulated condition compared with 18 and 12% for IMP321-stimulated CD8 cells. The same proliferative effect of IMP321 was also observed on CD4⁺ T cells but to a lesser extent (data not shown).

The IMP321-stimulated cells are Ag-experienced effector or effector memory CD8⁺ T cells

To further characterize the specific population of IMP321-induced IFN-γ⁺TNF-α⁺CD8⁺ T cells, PBMCs were analyzed at 18 h to discriminate naive, effector, effector memory, and central memory (CM) T cell subsets. Naive T cells were not stimulated by IMP321, as CD45RA⁺CD11a^dim, CD45RA⁺CD27⁺, or CD45RA⁺CCR7⁺ cells were underrepresented in the IMP321-induced IFN-γ⁺TNF-α⁺CD8⁺ T cell subset while representing a third of the unstimulated CD3⁺CD8⁺ population (Fig. 7⇓A). In contrast, effector T cells were stimulated by IMP321, as CD45RA⁺CD11a^high or CD45RA⁺CD27⁻ or CD45RA⁺CCR7⁻ cells were overrepresented in the IMP321-induced IFN-γ⁺TNF-α⁺CD8⁺ T cell subset compared with the unstimulated CD3⁺CD8⁺ population (Fig. 7⇓A).

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FIGURE 7.

The IMP321-stimulated cells are Ag-experienced effector or EM CD8⁺ T cells. PBMCs were incubated with IMP321 (1 μg/ml) for 18 h in the presence of brefeldin A for the last 16 h. Cells were stained with CD8-allophycocyanin-Cy7, CD45RA-PE-Cy7, and either CD11a-allophycocyanin, CD27-allophycocyanin, or CCR7-Alexa 647 as indicated, fixed, permeabilized, stained with CD3-PerCP-Cy5.5, anti-IFN-γ-PE, and TNF-α-FITC mAbs and analyzed by flow cytometry (A). The percentage of IFN-γ⁺/TNF-α⁺ in CD3⁺CD8⁺ population is shown in unstimulated (upper left panel) and stimulated conditions (upper right panel). The expression of CD45RA and either CD11a, CD27, or CCR7 were analyzed on total CD3⁺CD8⁺ in unstimulated condition (left panels) and on IFN-γ⁺/TNF-α⁺ CD3⁺CD8⁺ (right panels) in stimulated condition (A). PBMCs from 12 subjects were stimulated or not with IMP321 and stained with CD8-allophycocyanin-Cy7, CD45RA-PE-Cy7, anti-CCR7-Alexa 647 mAbs, fixed, permeabilized, and then stained with CD3-PerCP-Cy5.5, anti-IFN-γ-PE, and granzyme A- or B-FITC mAbs (B). PBMCs from six subjects were stimulated or not with IMP321 and stained with CD8-allophycocyanin-Cy7, CD45RA-PE-Cy7, anti-CCR7-Alexa 647 and CD27-PE (C) or CD28-PE mAbs (D), fixed, permeabilized, and then stained with CD3-PerCP-Cy5.5 and anti-IFN-γ-FITC mAbs. The percentage of the different populations based on the expression of CD45RA, CCR7, and granzyme (B), CD27 (C), or CD28 (D) in whole CD3⁺CD8⁺ population and in CD3⁺CD8⁺ expressing IFN-γ was determined in unstimulated (left panels) and IMP321-stimulated (right panels) conditions, respectively. The means of percentage of each population are presented on statistical views. M, memory; N, naive; E, effector.

We then analyzed the CD45RA/CCR7 phenotype in a larger cohort of 12 donors to present a more representative analysis of the percentages found in the different subgroups. CM phenotype CD45RA⁻CCR7⁺CD8⁺ T cells were not stimulated by IMP321 because this subset was less represented in the IMP321-induced IFN-γ⁺CD8⁺ T cells compared with the unstimulated control population (Fig. 7⇑, B–D). Effector memory (EM) CD45RA⁻CCR7⁻CD8⁺ T cells were found with similar frequencies in the IMP321-induced IFN-γ⁺ CD8⁺ T cell subset as in the unstimulated control population. Nonetheless, when refining the analysis further we found that virtually all IMP321-activated EM cells expressed granzymes A or B (Fig. 7⇑B) but not CD27 (Fig. 7⇑C) and CD28 (Fig. 7⇑D), in stark contrast to the control population. Similarly, IMP321-induced effector (E) IFN-γ⁺ CD8⁺ T cells also expressed granzymes A or B without CD27 and CD28 (Fig. 7⇑, B–D). Virtually, all CD8⁺ T cells responding to IMP321 by producing IFN-γ were granzymes A/B⁺CD27⁻CD28⁻ indicating their terminal effector differentiation phenotype.

IMP321 induces a full Tc1 activation but no IL-10 production, in contrast to TLR agonists

PBMCs from four donors were stimulated with IMP321 or with the following TLR agonists at their optimal concentrations: TLR1-2 (lipopeptide), TLR3 (dsRNA), TLR4 (LPS), TLR5 (flagellin), TLR6-2 (macrophage-activating lipopeptide-2), TLR7-8 (ssRNA), or TLR9 (CpG). At these concentrations, all the TLR agonists were found to be at least as potent as 1 μg/ml IMP321 at inducing high IFN-γ, TNF-α, IL-1β, IL-6, CCL4, CCL5, and CCL2 concentrations in 48-h supernatants (data not shown). The immunosuppressive cytokine, IL-10, was only induced by the TLR ligands (Fig. 8⇓A) but not by IMP321 (Fig. 2⇑ and four additional donors in Fig. 8⇓A). In fact, IMP321 even decreased the level of IL-10 secretion in some donors compared with the unstimulated condition (Figs. 2⇑ and 8⇓A).

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FIGURE 8.

IMP321 induces a full Tc1 activation because it does not induce IL-10 production, in contrast to TLR agonists. PBMCs were incubated with or without IMP321 (1 μg/ml) or TLR agonists (optimal concentrations): TLR1-2, TLR3, TLR4, TLR5, TLR6- 2, TLR7-8, or TLR9 agonists for 24 or 48 h. IL-10 was measured in supernatants by CBA assays. Data represent the concentrations of IL-10, expressed in picograms per 10⁶ cells on a logarithmic scale (A). PBMCs were stimulated or not with IMP321 (1 μg/ml) or TLR agonists (optimal concentrations) for 18 h. Cells were stained with fluorochrome-conjugated CD3, CD4, CD8, anti-IFN-γ, TNF-α, and IL-2 mAbs as described in Fig. 4⇑A. The percentage of CD3⁺CD8⁺ T lymphocytes expressing IFN-γ, TNF-α, and/or IL-2 was determined by flow cytometry. The percentages of IFN-γ⁺TNF-α⁻, IFN-γ⁻TNF- α⁺ and IFN-γ⁺TNF-α⁺ in CD3⁺CD8⁺ cells for four subjects are presented (B).

In line with this IL-10 induction, the TLR agonists were unable to induce a full Tc1 activation with no IFN-γ⁺CD8⁺ T cells being induced at 18 h, compared with IMP321 (Fig. 8⇑B). Only an elevated inflammatory background type of response (i.e., expression of TNF-α by some T cells) was induced by TLR agonists (Fig. 8⇑B). Similarly, IMP321, but not the TLR agonists, induces IFN-γ⁺TNF-α⁺ cells in the CD3⁻CD56⁺ NK subset (10 PBMC samples tested, data not shown). Overall, these data indicate that the lack of IL-10 induction by IMP321 correlates with its ability to induce IFN-γ⁺TNF-α⁺ Tc1-type CD8 and NK effector cells, in contrast to TLR agonists.