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Keywords:

  • small cell lung cancer;
  • Treg cell;
  • cytokine;
  • IL-15;
  • survival

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Small cell lung cancer (SCLC) kills at least one person every 2 hr in the United Kingdom. Some patients do relatively well but most have rapidly progressive disease. There is no effective treatment and overall 2-year survival is less than 5%. Patients with SCLC have poorly understood local and systemic immune defects and can be immunocompromised. As CD4+ T lymphocytes coordinate and regulate immunity, a better understanding of interactions between SCLC tumour cells and CD4+ T cells may lead to effective molecular immunotherapy. We show that some, but not all, SCLC tumour cell lines secrete molecules that induce IL-10 secretion by and de novo differentiation of functional CD4+CD25+FOXP3+CD127loHelios regulatory T (Treg) cells in healthy blood lymphocytes. FOXP3+ T cells were found in SCLC tumour biopsies, and patients with higher ratios of FOXP3+ cells in tumour infiltrates have a worse survival rate. The inhibitory effect of SCLC tumour cells was not affected by blocking IL-10 receptor or TGF-β signalling but was partially reversed by blocking IL-15, which is reported to be involved in human Treg cells induction. IL-15 was secreted by SCLC cells that inhibited CD4+ T-cell proliferation and was present in SCLC biopsy tumour cells. These novel findings demonstrate that SCLC tumour cells can induce CD4+ T-cell-mediated immunosuppression. This gives a potential mechanism by which SCLC tumour cells may downregulate local and systemic immune responses and contribute to poor patient survival. Our data suggest that IL-15 and Treg cells are potential new therapeutic targets to improve immune response and patient survival in SCLC.

Small cell lung cancer (SCLC), which constitutes 10–20% of all lung cancers, is particularly aggressive with wide-spread metastasis at presentation. At least one person dies of SCLC every 2 hr in the United Kingdom, and, despite treatment, 2-year survival is less than 5%.1, 2 Novel therapeutic strategies are urgently required. A minority of patients with SCLC mount immune responses to tumour-associated antigens and have a more favourable prognosis.3 Conversely, most patients with SCLC are immunocompromised with poorly understood local and systemic immune defects that correlate with worse morbidity and mortality.4, 5

The immune system protects the host from tumour development, growth and metastasis,6–8 and immune suppression9 (including that caused by HIV infection10) promotes lung cancer development. CD4+ T lymphocytes play a central role in anti-tumour immune responses.11 The CD4+ T-cell subset contains both ‘helper’ T cells, which coordinate acquired immune effector responses and generate cell-mediated and humoral effector immunity, and ‘regulatory’ T (Treg) cells, which downregulate immune effector responses.12 Treg cells can inhibit activation, expansion and effector function of other T cells.13 Two broad Treg subsets that express the transcription factor forkhead box protein P3 (FOXP3) are described in vivo: ‘natural’ Treg (nTreg) cells, which are released from the thymus, and ‘induced’ Treg (iTreg) cells, which are differentiated from resting CD4+ T cells as part of the response to antigen challenge. CD127, the α-chain of the IL-7 receptor, has been shown to be a marker to discriminate between human CD4+ regulatory and activated T cells, and its expression inversely correlates with FOXP3 and suppressive function.14, 15

Helios protein, a member of the Ikaros transcription factor family, has been reported to distinguish Treg subsets with nTreg cells being positive and iTreg cells being negative for Helios protein.16 However, Helios expression has been recently observed in peripherally induced FOXP3+ regulatory cells17 and is associated with T-cell activation and proliferation.18

The role of Treg cells in cancer is not fully understood and differs with the type of tumour. FOXP3+ T cells were found to infiltrate human non-SCLC and ovarian cancer19 and have been subsequently identified in a number of cancers. However, the effect of FOXP3+ T-lymphocyte infiltration on patient survival is controversial and has been variously reported to have no prognostic effect,20, 21 to be associated with improved survival13, 22, 23 or to predict poor survival.24, 25 Understanding how Treg cells contribute to cancer progression may need to be determined on a tumour-type basis.

Progress in understanding SCLC pathobiology has been hampered by lack of animal models. To determine how SCLC tumour cells can affect immune system responses, we investigated the effects of tumour cell lines on CD4+ T cells isolated from healthy human blood and studied SCLC biopsies and matching clinical data from a cohort of 65 patients. Here, we show for the first time that some, but not all, SCLC tumour cell lines constitutively secrete molecules that suppress proliferation of activated CD4+ T cells from healthy donors in vitro by inducing de novo CD4+CD25+ FOXP3+CD127loHelios Treg cell differentiation. The suppressive effect is independent of IL-10R or TGF-β signalling but can be partially reversed by IL-15 blockade. IL-15 is secreted by those SCLC tumour cells that induce Treg cells. IL-15 is present in tumour cells in SCLC biopsies, and patients with higher ratios of FOXP3+ cells in the immune infiltrate of their tumours have a significantly worse survival rate of 1,000 days post-diagnosis. These data indicate that IL-15 and Treg cell function may be new targets for therapy to improve immunosuppression and survival in SCLC.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Ethical approval

The use of healthy blood donors and of patient material and the clinical data were approved by the Lothian Research Ethics committee.

SCLC cells

Mycoplasma-free cell banks of SCLC cell lines, NCI-H69, NCI-H345 (ECACC, Health Protection Agency, Porton Down, UK) and NCI-H510 (ATCC, LGC Standards, Teddington, UK) were grown and stored in liquid nitrogen. Cells in suspension at 2–5 × 105 ml−1 were passaged up to ten times in 75-cm2 flasks (Sigma, Poole, UK) in complete RPMI (RPMI1640 medium supplemented with 10% heat-inactivated fetal calf serum, 5 μg/ml L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin; all obtained from Invitrogen, Paisley, UK) at 37°C in a humidified 5% CO2 incubator.

H69 SCLC cell conditioned medium and cell lysate

H69 cells were washed and resuspended at 2.5 × 106 ml−1 in 10-ml serum-free Iscove's modified Dulbecco's medium (IMDM; Sigma) supplemented with 50 U/ml penicillin and 50 μg/ml streptomycin in 25-cm2 tissue culture flasks (Sigma) for 72 hr. Conditioned medium (CM) was centrifuged at 1,000g for 5 min to remove cells. To lyse cells, the pellet was resuspended in lysis buffer [10 ml phosphate buffered saline (PBS), 1 Mimi protease inhibitor cocktail tablet (Roche, Welwyn Garden City, UK), 1 mM Na3VO4 and 100 μl octylphenoxypolyethoxyethanol (IGEPAL, Sigma)] for 30 min on ice.

Isolation of peripheral blood mononuclear cells

Human peripheral blood from normal healthy donors was layered onto Lymphoprep™ (Axis-Shield, Cambridgeshire, UK) and centrifuged at 2,000g for 20 min. Cells from the interface were washed thrice with PBS by centrifugation at 1,000g to remove platelets.

Mixed lymphocyte reactions

Two-way mixed lymphocyte reactions (MLRs) were established from peripheral blood mononuclear cells (PBMCs) of unrelated donors. H69 SCLC cells were washed, resuspended in PBS (106 cells per milliliter), incubated with mitomycin-C (50 μg/ml; Sigma) for 3 hr at 37°C, washed thrice in PBS and resuspended in complete IMDM (10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin). In a 96-well plate (Sigma), 5 × 104 PBMCs per donor per well were combined with mitomycin-C-treated SCLC cells at a 1:1 ratio in 200 μl complete IMDM at 37°C in 5% CO2 in a humidified incubator for 72 hr.

Naïve/CD4+ T-cell activation

CD4+ T cells were isolated from PBMCs by negative selection on MACS™ columns using the CD4+ T-cell isolation kit (Miltenyi Biotec, Bisley, UK). Purity was 95.6% ± 0.9%, which was evaluated by flow cytometry. Naïve CD4+ T cells were purified from PBMCs using naïve T-cell isolation kit II (Miltenyi Biotec). The purity of CD4+CD45RA+ cells was above 95%. Purified naïve/CD4+ T cells, at 2 × 105 cells per well, were stimulated with 1.25 μg per well immobilised anti-CD3 (OKT3, pre-coated for 6 hr at 37°C) and 1 μg/ml soluble anti-CD28 (CD28.2) monoclonal antibodies (e-Bioscience, Hatfield, UK) or with Dynabeads CD3/CD28 T-cell expander (one bead per cell, Invitrogen) in 1 ml per well complete IMDM in 24-well plates (Millipore, Watford, UK) at 37°C in 5% CO2 in a humidified incubator for 72 hr.

Cell proliferation

Carboxyfluorescein diacetate succinimidyl ester incorporation

Cells were incubated with carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen; 1 μM; Molecular Probes Invitrogen) according to manufacturer's instructions. After 72 hr, cells were harvested, and cell division was estimated by flow cytometry. Data were acquired on a FACSCalibur (BD Biosciences) flow cytometer and analysed with FlowJo™ software.

3H-Thymidine incorporation

Cells were cultured for 48 hr and then pulsed for 24 hr with 1 μCi 3H-thymidine (Perkin Elmer, Cambridge, UK) per well. Cells were harvested with a Tomtec (Leamington Spa, UK) cell harvester and 3H-thymidine incorporation counted in a β-plate reader (Wallac, UK).

Cytokine ELISAs

IL-10, IFN-γ, IL-4 and IL-17 were measured by Duo-set ELISAs (R&D systems, Abingdon, UK) and IL-15 by Ready-SET-Go!® (e-Bioscience) according to manufacturers' instructions. Plates were read at 450 nm (650 nm reference) on a Biotek Synergy HT plate reader (Fisher Scientific, Loughborough, UK).

Coculture of SCLC cells and CD4+ T cells

SCLC cells were treated with mitomycin-C as above. In 96-well plates, CD4+ T cells (2 × 105 cells per well) were activated with immobilised anti-CD3 (0.5 μg per well) and soluble anti-CD28 (0.1 μg per well) and cultured alone or in combination with mitomycin-C-treated SCLC cells at a 1:1 ratio.

Transwell cocultures

Polycarbonate 24-well transwell inserts (0.4 μM) and receiver trays (Millipore) were used to physically separate naïve/CD4+ T cells and SCLC cells. Naïve/CD4+ T cells (2 × 105) in the lower chambers were stimulated with immobilised anti-CD3 and soluble anti-CD28 monoclonal antibodies in 0.8 ml complete IMDM. Complete IMDM (0.2 ml) only or containing SCLC cells at T cell/SCLC cell ratios of 1:1, 1:2 or 1:4 was added to the upper chambers.

Treg functional assay

CD4+ T cells isolated from single donors were divided into two aliquots. One aliquot was stimulated as above with anti-CD3/CD28 antibodies and cultured alone or cocultured with SCLC cells at 1:4 ratio in transwells for 3 days. The second aliquot was immediately frozen and stored at −80°C for 3 days in freezing medium (40% complete IMDM with 50% FBS and 10% DMSO; Sigma).

On Day 3, frozen naïve/CD4+ T cells were quickly thawed (37°C, 5 min) and washed by centrifugation in warm complete 3× IMDM. Viable cells (>95%) were counted by trypan blue exclusion and labelled with CFSE. The previously cultured autologous CD4+ T cells were harvested and counted. CFSE-labelled defrosted CD4+ T cells (105 cells per well) and the unlabelled cultured autologous CD4+ T cells (2 × 105 cells per well) were cocultured in 24-well plates in the presence of Dynabeads™ CD3/CD28 T-cell expander (Invitrogen). After 72-hr proliferation, cell division of CFSE-labelled CD4+ T cells was analysed by flow cytometry.

Cytokines and cytokine-signalling blockers

IL-15 (20 ng/ml; R&D) was added as indicated. TGF-β signalling was blocked with monoclonal neutralising anti-TGF-β antibody (active against all isoforms) and compared to isotype control antibody at 10 μg/ml (R&D Systems, Abingdon, UK) or with 1 μM of the TGF-β and activin-signalling inhibitor SB 431542 hydrate (Sigma) and vehicle control. IL-10 signalling was blocked using monoclonal blocking anti-human IL-10 Rα antibody compared to isotype control antibody (20 μg/ml; R&D). IL-15 was blocked using monoclonal neutralising anti-IL-15 compared to isotype control antibody (e-Bioscience).

Flow cytometric analysis of protein expression

Surface marker staining used standard protocols with mouse monoclonal antibodies: FITC-anti-CD8, RPE-anti-CD4; RPE-Cy5-anti-CD3 and relevant isotype controls (all obtained from DAKO, UK); PE-anti-CD45RA (HI100) and PE-anti-CD127 (eBioRDR5; both obtained from e-Bioscience). For Treg cell (CD4+CD25+FOXP3+) staining, the human Treg cell detection kit (Miltenyi Biotec) was used. Briefly, CD4+ T cells were stained with FITC-anti-CD4 (VIT4) and PE-anti-CD25 (4E3), fixed and permeabilised, blocked with FcR blocking reagent and stained with APC-anti-FOXP3 (3G3) or isotype control. Treg cell was also stained with FITC-anti-CD4 (VIT4), PE-anti-CD127 (eBioRDR5) and APC-anti-FOXP3 (3G3). In some experiments, freshly isolated CD4+ T cells were labelled with CFSE as above, cocultured in transwells with H69 cells 1:4 for 3 days, then fixed and permeabilised, blocked with FcR blocking reagent, stained with PE-anti-mouse/human Helios protein (22F6; Biolegend, UK) and APC-anti-human FOXP3 (3G3) or isotype control and analysed by Flow Cytometry. Data were acquired on a FACSCalibur (BD Biosciences) flow cytometer and analysed with FlowJo™ software.

Immunofluorescence and immunohistochemistry

Paraffin-embedded formalin-fixed lung tumour biopsies from patients with SCLC were obtained from the Department of Pathology, Royal Infirmary of Edinburgh. Patient details and survival data were collected by P.H. Sixty-five patients (26F, 39M; median age 66 years) were studied; median survival time was 235 days.

Sections (3 μm) were antigen retrieved in BORG Decloaker pH9.5 (1,000 W microwave, 10 min maximum power; Biocare Medical, Walnut Creek, CA). For immunofluorescence, the sections were blocked (5% goat serum, 30 min), incubated with rabbit-anti-CD3 (DAKO) and monoclonal mouse-anti-FOXP3 (e-Bioscience) diluted in 5% goat serum for 1 hr, washed and incubated with Alexa-488-goat anti-rabbit IgG and Alexa-568-goat anti-mouse IgG (both 1:1000, Invitrogen) for 30 min followed by 4,6-diamidino-2-phenylindole (DAPI, 0.1 μg/ml) to stain nuclei. The sections were mounted in aqueous mounting medium (DAKO) and analysed on a Leica TCS SP5 confocal microscope.

For immunohistochemistry, the sections (3 μm) were antigen retrieved as above, blocked with 3% hydrogen peroxide for 15 min (Sigma), washed with TBS, loaded into a Shandon Sequenza® slide rack (Fisher Scientific, Loughborough UK) and 100 μl primary antibody (mouse monoclonal anti-CD3, anti-CD4, anti-CD45; Novocastra Reagents, Leica Microsystems, Wetzlar, Germany) or anti-IL15 (Abcam, Cambridge, UK) added to each slide overnight at 4°C. Slides were rinsed twice with TBS and EnVision™ developing reagents (DAKO, Ely, UK) used as per manufacturer's instructions. Slides were washed with TBS, and 100 μl 3,3′-Diaminobenzidine (DAB, DAKO) solution was applied for 5 min. Cytospins of H69 and H510 cells were air dried and fixed in 90% anhydrous acetone/10% methanol. IL-15 immunohistochemistry was performed on cytospins as above but without antigen retrieval.

Statistics

Data are expressed as mean ± SEM. The statistical significance of the difference between two groups was performed using Student's t-test. For multiple group comparisons, one-way ANOVA with Tukey's post-test was performed. Correlation between values was evaluated using non-parametric Spearman's rank correlation. Kaplan–Meyer survival curves were plotted, and all statistical analysis was performed using GraphPad Prism™ 5 (Graph Software, San Diego, CA). Probability values (p) <0.05 were considered statistically significant.

Microarray analysis

All comparative analysis was carried out by the Bioinformatics Team in the BHF Centre for Research Excellence, University of Edinburgh. Expression data were downloaded from the Gene Expression Omnibus. All result sets were derived from variants of the Affymetrix Human Genome U133 GeneChip. Expression values were converted to the linear scale where appropriate. Annotation for the probe identifiers on these chips was derived from appropriate annotation packages provided by the Bioconductor software suite (www.bioconductor.org). All data were output to a relational database and examined by use of a web-based query builder. Although the vast majority of probe identifiers were comparable between chips, this web interface allowed qualitative comparison of expression for different probe IDs mapping to the same gene via their gene IDs from Entrez (cite entrez gene PMID 15608257). Because these data were derived from disparate experiments and not cross-normalised, this methodology allows only for a coarse comparison of expression levels.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

SCLC-secreted soluble molecules inhibit CD4+ T-cell proliferation through preventing activated CD4+ T cells from entering cell cycle

Three SCLC tumour cell lines were tested to evaluate the effects on lymphocyte proliferation. Coculture of two-way PBMC MLRs (driven by recognition of disparate MHC-II antigens on antigen-presenting cells, which are recognised by CD4+ T lymphocytes) with non-dividing (mitomycin-C treated) H69 and H345 significantly reduced proliferation, whereas H510 did not (Fig. 1a).

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Figure 1. H69 SCLC cells and their soluble molecules inhibit healthy blood-derived CD4+ T-cell proliferation and induce CD4+CD25+FOXP3+CD127lo Treg cells. (a) MLR inhibition by mitomycin-C-treated H69 and H345 cells. Mean ± SEM of seven experiments. *p < 0.05, ***p < 0.0001. (b) CD4+ T-cells activated by CD3/28 ligation in the presence of different ratios of mitomycin-C-treated H69 cells. (c) CD4+ T cells activated by CD3/28 ligation in the presence of H69, H510 and H345 cells at 1:4 ratio. (d) H69 cells separated from CD4+ T cells in transwell cocultures inhibit anti-CD3/CD28-induced 3H-thymidine incorporation; mean ± SEM of eight donors; ***p < 0.0001. (e) H69 CM reduces CD4+ T-cell proliferation induced by CD3/CD28 ligation; mean ± SEM of seven donors; ***p < 0.0001. (f) Representative flow cytometry showing that transwell coculture at 1:4 CD4+ T cells:SCLC cells reduces percent of cells undergoing division and number of division cycles completed by CFSE-labelled CD4+ T cells. (g) Representative flow cytometry showing increased Treg cells in purified CD4+ T cells activated in transwell cultures for 72 hr by CD3/CD28 ligation alone or with different ratios of H69 SCLC cells; cells were stained with anti-CD4, anti-CD25 and anti-FOXP3 antibodies and gated on CD25 expression. (h) CD4+ T cells cocultured with H69 (1:4 ratio of CD4+:H69) stained with anti-CD4, anti-CD25, anti-CD127 and anti-FOXP3. (i) Data from six donors cultured as in (a) showing increased percent of CD25+FOXP3+ cells in the CD4+ gate with increasing numbers of H69 cells. **p < 0.01, ***p < 0.0001. (j) Data from four donors showing that CD3/CD28 ligation in 40% H69 CM increases Treg cells differentiation; **p < 0.01.

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To investigate the direct effects on CD4+ T lymphocytes, negatively selected, purified CD4+ T-cells from healthy donors were cultured alone or with 1:1 mitomycin-C-treated H69 cells and activated by ligation of CD3 and CD28. Proliferation was significantly suppressed by mitomycin-C-treated H69 cells after 72 hr in a dose-dependent fashion (Fig. 1b). To determine whether the different effects between the SCLC cell lines on MLR proliferation were the same in this system of direct CD4+ T cell activation, the cell lines were compared. Mitomycin-C-treated H69 and H345 cells inhibited proliferation, but H510 had no effect (Fig. 1c). To determine whether or not the inhibition required cell–cell contact, CD4+ T cells were stimulated for 72 hr by CD3/CD28 ligation in the bottom of transwells with medium only or with SCLC cells in the upper wells. CD4+ T-cell proliferation was inhibited by H69 cells in a dose-dependent fashion (Fig. 1d), indicating that cell–cell contact was not necessary. To assess whether the soluble inhibitory factor was constitutively produced, serum-free CM from H69 or H510 cells was added to cultures of CD4+ T cells stimulated for 72 hr by CD3/CD28 ligation. Proliferation was reduced by the addition of 20% H69-CM but not by 20% H510-CM (Fig. 1e).

To determine the nature of the proliferation block, CD4+ T cells were labelled with CFSE immediately after purification and stimulated by CD3/CD28 ligation in transwells with SCLC cells at a ratio of 1:4. When cells divide, exactly half of the fluorescent CFSE is transferred to daughter cells allowing estimation of divisions by flow cytometry. Figure 1f shows that both the fraction of dividing CD4+ cells and the number of divisions per cell were significantly reduced.

SCLC-secreted soluble molecules induce de novo functional CD4+CD25+FOXP3+CD127loHelios Treg cells

To investigate whether decreased proliferation was due to Treg cell induction, purified CD4+ T cells were activated by CD3/CD28 ligation and cultured alone or with H69 SCLC tumour cells in transwells or in H69 CM for 72 hr, stained and analysed by flow cytometry. The population of Treg cells (CD4+CD25+FOXP3+CD127lo) was increased in transwell cocultures (Figs. 1g1i) and by H69 CM (Fig. 1j) in a dose-dependent fashion.

To determine whether increased Treg cells was due to expansion of preexisting cells (nTreg cells) or de novo differentiation of naïve cells (iTreg cells), first, CFSE-labelled CD4+ T cells were activated as above in transwell cocultures (1:4 of CD4+:H69 cells) for 72 hr. H69 cells increased differentiation of FOXP3+Helios cells despite reduced proliferation, and the ratio of FOXP3+Helios/FOXP3+Helio+ cells increased (Fig. 2a). Importantly, Treg cells (CD4+CD25+ FOXP3+CD127lo) induction is observed from naïve T cells with a phenotype of CD4+CD45RA+CD25FOXP3CD127hi (Fig. 2b), suggesting that the Treg phenotype was induced by differentiation of naïve T cells rather than by expansion of preexisting nTreg cells.

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Figure 2. The induced Treg cells were derived from differentiation of naïve T cells and were functional suppressive. (a) Representative flow cytometry of purified CD4+ T cells in transwell cultures activated as in Fig. 1g with 1:4 CD4:H69 cells showing increased FOXP3+ cells despite reduced cell proliferation. The increased FOXP3+ cells are Helios. (b) Purified naïve T cells and the cultured cells as in (a) stained with anti-CD4, anti-CD45RA, anti-CD25, anti-CD127 and anti-FOXP3. Representative flow cytometry showing H69 cell-induced FOXP3+CD127lo Treg cells population from naïve T cells. (c) Representative flow cytometry showing that unlabelled cells previously activated in transwell cultures for 72 hr by CD3/CD28 ligation with H69 SCLC cells at 1:4 T:SCLC ratio inhibit CD3/CD28 ligation-induced proliferation of autologous CFSE-labelled CD4+ T cells. (d) Percent of activated CD4+ T cells undergoing division is reduced when cocultured with autologous unlabelled cells previously activated with H69 SCLC cells in transwells as in (e); mean ± SEM of three donors. *p < 0.05.

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To determine whether Treg cells were functional, CD3/CD28-activated CD4+ T cells previously cocultured with SCLC cells (ratio: 1:4) in transwells for 3 days were harvested and cocultured with CFSE-labelled, autologous, unstimulated T cells in the presence of anti-CD3/CD28 beads for 72 hr. Proliferation of CFSE-labelled cells was significantly suppressed by the presence of unlabelled CD4+ T cells previously cocultured with SCLC cells (Figs. 2c and 2d).

The suppressive effect is not dependent on IL-10 or TGF-β

To examine whether proliferation inhibition was associated with immunosuppressive cytokines, levels of IL-10 and TGF-β were determined in supernatants of CD4+ T cells activated by CD3/CD28 ligation in transwell coculture or in H69 SCLC CM. No TGF-β (activated or latent) was detected above background, and neither antibody-mediated inhibition nor pharmacological inhibition of TGF-β signalling had any effect on the inhibition of proliferation (data not shown). IL-10 secretion was significantly increased in transwell cocultures (Fig. 3a) and in separate experiments by H69 CM (Fig. 3b) in a dose-dependent fashion. However, the addition of anti-IL-10R-blocking antibody failed to reverse the inhibition in both transwell (Fig. 3c) and CM experiments (Fig. 3d).

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Figure 3. SCLC-induced inhibition of proliferation is IL-10 independent although secretion of IL-10 is increased. (a and b) CD4+ T-cell IL-10 production after 72 hr CD3/CD28 ligation in (a) transwell cocultures or (b) H69 CM; mean ± SEM of six donors; *p < 0.05, **p < 0.01, ***p < 0.0001. (c and d) Proliferation of CD4+ T cells after 72 hr CD3/CD28 ligation in (c) transwell cocultures or (d) H69 CM in the presence of anti-IL-10 neutralising antibody or isotype control; mean ± SEM of three donors; *p < 0.05, ***p < 0.0001.

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IL-15 is produced by H69 SCLC cells and contributes to the induction of functional iTreg cells

To determine which components of CM inhibited CD4+ T-cell proliferation, differential column filtration was carried out. The molecules involved were <30 kDa (Fig. 4a), which suggested that soluble cytokines might be candidates. As H510 SCLC tumour cells did not suppress T-lymphocyte proliferation (Fig. 1a), we compared publicly available microarray data on cytokine gene expression in H69 cells, which inhibited proliferation relative to H510 cells which did not. This identified a number of cytokine genes (IL-1a, IL-11, IL-15, IL-16, BMP-7, CSF-2 and TGF-β2) upregulated in H69 cells. As we had ruled out TGF-β signalling, of the remainder, IL-15 was of particular interest for further investigation, as this has previously been shown to be involved in Treg cells induction.26, 27 We found that H69 cell lysates contained much higher levels of IL-15 protein than those from H510 cells (Fig. 4b). Anti-IL-15 immunostaining was present in H69 but not in H510 SCLC cells (Fig. 5a). Blocking IL-15 activity partially reversed H69 CM-induced inhibition of proliferation (Fig. 4c) and reduced CD4+ T-cell secretion of IL-10 (Fig. 4d). Importantly, IL-15 increased FOXP3 population from naïve CD4+ T cells activated by CD3/CD28 ligation in the context of H69 cells (Fig. 4e).

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Figure 4. IL-15 is produced by H69 but not H510 SCLC cells and contributes to suppression of CD4+ T-cell proliferation and increased IL-10 secretion induced by H69 CM. (a) The soluble activity in H69 CM responsible for inhibition of CD4+ T-cell proliferation is >30 kDa; mean ± SEM of three donors; *p < 0.05. (b) IL-15 is present in CM and lysate of H69 cells. (c) Data from eight donors showing that IL-15-neutralising antibody added to CD4+ T cells activated by CD3/CD28 ligation in 40% CM derived from H69 SCLC cells partially reverses the inhibition of proliferation; **p < 0.01. (d) Data from six donors showing decreased IL-10 production by CD4+ T cells activated by CD3/CD28 ligation in 40% CM derived from H69 SCLC cells in the presence of IL-15-neutralising antibody or isotype control; *p = 0.0313. (e) Representative flow cytometry showing Treg cells induced from naïve CD4+ T cells activated by CD3/CD28 ligation for 72 hr alone in the presence of IL-15 or with H69 cells at 1:4 ratio of T:H69 in transwell cultures; cells were stained with anti-CD4, anti-CD25 and anti-FOXP3 antibodies and gated on CD25 expression.

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Figure 5. SCLC tumour cells express IL-15 and are infiltrated by Treg cells. (a) Immunohistochemistry showing IL-15 inside H69 but not H510 SCLC tumour cell lines. (bd) Immunohistochemistry of representative SCLC biopsy sections showing (b) IL-15 inside tumour cells, (c) CD3+ T cells and (d) CD4+ T cells. (eh) Immunofluorescence showing SCLC section simultaneously stained for (e) CD3+ T cells, (f) FOXP3 transcription factor, (g) all nucleated cells (DAPI) and (H) merged stains and high power of merged stains showing the presence of CD3+ T cells double stained for FOXP3. (ik) Immunohistochemistry stained for (i) FOXP3 transcription factor, (j) CD45 leukocytes and (k) negative control.

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Tumour cells from patients with SCLC contain IL-15, and biopsies are infiltrated by Treg cells

In view of these results, we investigated IL-15 and Treg cell presence in biopsies from patients with SCLC. Critically, malignant cells in tumour biopsies contain IL-15 protein (Fig. 5b). Although considerably fewer in number than tumour cells, CD3+ and CD4+ T cells were present within the stroma of SCLC tumours (Figs. 5c and 5d). Immunofluorescence (Figs. 5e5h) demonstrated that some CD3+ T cells, but none of the tumour cells, were also positive for nuclear FOXP3.

Increased proportion of Treg cells in SCLC tumour biopsy infiltrates negatively correlates with patient survival

We examined the relationship between Treg cells in leukocyte infiltrates of SCLC tumour biopsies and patient survival retrospectively in 65 ‘typical’ cases of SCLC. As we had shown that no tumour cells expressed FOXP3, we stained parallel sections from the biopsies with anti-FOXP3 (nuclear staining; Fig. 5i) and anti-CD45 (pan-leukocyte marker; Fig. 5j). Sections were counted blindly under 400× magnification to determine the ratio of FOXP3+:CD45+ cells as a measure of proportion of Treg cells in the infiltrating leukocyte population. Ten random high-power fields were counted per stain per biopsy, and the score per field was averaged. The number of FOXP3+ cells counted per field ranged from 1 to 40, median 8 per high-power field; the number of CD45+ leukocytes counted per field ranged from 3 to 127, median 37 per high-power field. The ratio of FOXP3+:CD45+ per field ranged from 1:33 to 1:1.25, median 1:3.7.

The effect on patient survival time was determined using Kaplan–Meier analysis. Critically, patients with FOXP3+:CD45+ ratios above the median value had significantly worse survival at 1,000 days post-diagnosis with a median survival time of 120 days compared to a median survival time of 410 days in the group with fewer FOXP3+ cells in their leukocyte infiltrates (Fig. 6).

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Figure 6. Poor patient survival correlates with a high intratumoural Treg cells:leukocyte ratio. Survival curve showing that for 65 patients diagnosed with SCLC, poor survival time correlated with having a high number of Treg cells in the intratumoural leukocyte infiltrate.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Immunotherapy for solid tumours has focussed on amplification of intratumoural natural killer (NK) cells, natural killer T (NKT) cells and CD8+ T-lymphocyte effector cell responses with limited success.28–31 Evidence suggests that CD4+ T cells, which are essential for generation of effective acquired immune responses to pathogens, are also important for anti-tumour immunity.11

We assessed whether SCLC tumour cells could modulate responses of CD4+ T cells from healthy donors. We show that some SCLC tumour cell lines (H69 and H345 but not H510) inhibit proliferation of activated CD4+ T cells. The inhibition induced by H69 cells was dose dependent, did not require cell–cell contact and was accompanied by functional CD4+CD25+FOXP3+CD127loHelios de novo Treg cell differentiation and IL-10 secretion. The inhibiting activity secreted by H69 cells had a molecular weight <30 kDa, which suggested that cytokines might be candidate molecules. Through interrogation of publicly available microarray data, we found that several cytokine genes were upregulated in H69 cells. These included IL-15, which is known to be involved in Treg cell induction.26, 27, 32 The H69-induced inhibition of healthy CD4+ T-cell proliferation was independent of IL10R and TGF-β-mediated signalling but was partially due to IL-15. IL-15 protein was present in H69, but not in H510 cells, and was found in tumour cells of SCLC biopsies.

IL-15 (MW = 14 kDa) belongs to the common gamma-chain (γc) cytokine family, is secreted by a variety of cell types,33, 34 binds to CD2533 and modulates the biology of NK cells, NKT cells, CD8+ T-cells, memory T-cells, monocytes and macrophages.35, 36 For these reasons, IL-15 has been promoted as a potential anti-tumour therapy to boost immune responses against cancer cells.37 Our data suggest that IL-15 may have other effects in SCLC where it may potentiate the observed immune suppression associated with the disease by promoting Treg cells induction.

We investigated the relevance of our results to survival in SCLC. An excess of Treg cells may lead to a failure of tumour immunosurveillance and contribute to progression. FOXP3+ Treg cells have been identified in a number of tumours.19–25, 38 However, the effect of FOXP3+ T-lymphocyte infiltration on patient survival is controversial and appears to be tumour dependent. FOXP3+ cell infiltration has been reported to have no prognostic effect in cutaneous malignant melanoma20 or prostate cancer,21 to be associated with improved survival in urinary bladder22 and colon cancers23, 39 and with poor survival in uveal melanoma24 and gastric cancer.25 These discordant effects suggest that tumour types must be investigated critically rather than ascribing a generalised function to Treg cells in cancer. We analysed 65 archival biopsies from patients with typical SCLC for which clinical details were available. To eliminate that the number of FOXP3+ cells present in a tumour was simply a reflection of overall leukocyte infiltration, the ratio of FOXP3+ cells in the leukocyte infiltrate was determined by staining parallel sections of needle lung biopsies from SCLC tumours for CD45, which marks all leukocytes, and for FOXP3. The results showed that a ratio of FOXP3+ cells in the leukocyte infiltrate above the median value predicted poor survival. There was no difference in age, gender, disease stage or treatments between the groups (data not shown), suggesting that the ratio of FOXP3+ cells in the leukocyte infiltrate can be regarded as an independent prognostic indicator in SCLC. Taken together, these results suggest that Treg cells and IL-15 are potential new therapeutic targets that may improve survival in this aggressive lung cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Mr. R. Morris and his team for histology support and the Bioinformatics Team in the BHF Centre for Research Excellence, University of Edinburgh, for the microarray analysis.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References