The authors have declared that no competing interests exist.
Obtained funding: SP D. Basso. Conceived and designed the experiments: D. Basso PF. Performed the experiments: PF EF CS DT SM D. Bozzato M. Pelloso GDF C-FZ FN CP CF E. Greco ST E. Gnatta M. Facco. Analyzed the data: D. Basso PF EF AP. Contributed reagents/materials/analysis tools: GB GS SP PP M. Plebani. Wrote the paper: D. Basso PF M. Falconi.
Current address: Department of Surgery, Università Politecnica delle Marche, Ancona, Italy
Blood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied
103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8+ lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced
In PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.
Metastases to distant organs, invasion of adjacent organs and angioinvasion characterize exocrine and endocrine pancreatic tumors
Inadequate immune response to cancer cells, a widely debated issue phenomenon, may enable primary tumor growth and metastasis
Immature myeloid cells may significantly contribute to tumor progression by inhibiting the adaptive immune response against tumor cells in lymphoid organs, and by migrating to tumor sites where they differentiate into highly immune suppressive tumor associated macrophages
Besides arginase and iNOS, tumor-induced MDSCs might overexpress HIF-1α, STAT3, C/EBPβ
The main aim of the present study was to contribute to the knowledge on immune suppression in human pancreatic benign and malignant diseases by studying the pattern of circulating and splenic lymphocyte subsets and immature myeloid cells in patients with PDAC, PDAC precursor lesions or pancreatic neuroendocrine tumors (NETs), using subjects with benign cystic adenoma and chronic pancreatitis as controls. Further endpoints were to assess whether pancreatic cancer cells are able to expand MDSCs
One-hundred-three consecutive patients (51 males, 52 females, median age: 62 years; age range: 21–83 years) were enrolled in two surgical units for pancreatic diseases (Department of Surgical, Oncological and Gastroenterological Sciences, DiSCOG, University of Padova and Department of Surgery, Chirurgia B, University of Verona, Italy) from November 2009 to April 2012. The study series included patients who underwent i) pancreatoduodenectomy or distal spleno-pancreatectomy for benign or malignant pancreatic disease; ii) splenectomy for non-neoplastic disease. Patients’ diagnoses and respective surgery are listed in
Diagnoses | Cases | Age | Surgery | Blood | Spleen | ||||
(M:F) | (range) | PD | DP | PR | T cells | M cells | T cells | M cells | |
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52 (31∶21) | 70 (48–83) | 19 | 21 | 12 | 51 | 34 | 20 | 16 |
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10 (5∶5) | 53 (34–76) | 0 | 10 | 0 | 10 | 5 | 10 | 8 |
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10 (2∶8) | 49 (21–73) | 2 | 7 | 1 | 10 | 7 | 8 | 8 |
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9 (1∶8) | 57 (30–83) | 1 | 7 |
1 | 9 | 0 | 5 | 0 |
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7 (5∶2) | 52 (25–81) | 2 | 2 | 3 | 7 | 4 | 3 | 2 |
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9 (4∶5) | 67 (54–75) | 6 | 3 | 0 | 9 | 0 | 0 | 0 |
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6 (3∶3) | 56 (36–76) | 6 splenectomies | 6 | 5 | 5 | 5 |
The total number of cases (Cases), the male:female (M:F) ratio, the mean age (years) with minimum and maximum values (range) of patients subdivided according to the histologically confirmed diagnoses, are reported in the first three columns. Surgery indicates the number of cases subjected to pancreatoduodenectomy (PD), distal pancreatectomy (DP) palliative resection (PR). Blood and spleen columns report the number of cases for whom T cells or immature myeloid cells (M cells) subsets were available. PDAC = pancreatic ductal adenocarcinoma; NETs = pancreatic neuroendocrine tumors; BPNs = pancreatic borderline neoplasms; SCA = serous cystadenoma; ChrPa = chronic pancreatitis;
Other tumors included 3 papillary, 3 duodenal and 3 stromal tumors.
2/7 patients underwent middle pancreatectomies.
Based on the absence or presence of vascular invasion, PDAC patients were divided into two groups: 28 (stage Ia = 1 case, stage Ib = 1, stage IIa = 10 and stage IIb = 16) without and 24 (stage III = 15 cases and stage IV = 9) with vascular invasion.
All patients gave their fully informed consent in writing to participate in the study, which was approved by the local ethics committee (Comitato Etico per la Sperimentazione of the University-Hospital of Padova; Prot. n° 1786P).
Before surgery and after overnight fasting, a potassium-EDTA blood sample was collected from each patient. Splenic tissue samples were obtained from patients who underwent distal splenopancreatectomy or splenectomy. After surgical removal, splenic samples (2.5×2.5×1 cm) were crushed and passed through a 100 µm cell strainer (BD Bioscience, San Josè, CA, USA). To obtain single cell suspensions, all samples were repeatedly passed through an 18G syringe and filtered through a 30 µm cell strainer (Partec, Munster, Germany), and then suspended in RPMI 1640 - FCS 10% (Invitrogen, Carlsbad, CA, USA ) for flow cytometry analysis.
Flow cytometry data from peripheral blood and splenic cell suspension, acquired using multicolor argon (488 nm) and helium-neon (633 nm) laser cytomics (FC 500 flow cytometer), were analyzed with CXP 2.2 software (Beckman Coulter, Miami, FL, USA). The following monoclonal antibodies were used: CD3-PC5, CD3-ECD, CD4-PE, CD8-ECD, CD45-FITC, CD45-ECD, HLA-DR-PC5, CD33-FITC and CD14-PC7 (Beckman Coulter, Miami, FL, USA); CD25-PE, CD4-FITC, CTLA4-PE (BD Biosciences); CD45-PC5 (Invitrogen); PDL1-PE (eBioscience, San Diego, CA, USA). The antibody panels were: CD4, CD8, CD3, CD45 for CD4+ or CD8+ T cells; CD4, CD3, CD25, CD45 for CD4+CD25+ lymphocytes; CD33, HLA-DR, CD45, CD14 for immature myeloid cells; CD33, HLA-DR, CD45, CD14, PDL-1 or CTLA4 for negative co-stimulatory molecules.
S100A8 and S100A9 mRNA was submitted for relative quantification by means of comparative CT method. Three micrograms of total RNA (High Pure RNA isolation kit, Roche, Monza, Italy) obtained from 9 PDAC splenic samples and from peripheral blood mononuclear cells (PBMC) of healthy blood donors, were used. S100A8 was amplified as described previously
The human BxPC3 (kindly donated by Dr. Andrea Galli, University of Florence, Italy), Capan1 and MiaPaCa2 (American Type Culture Collection) pancreatic cancer cell lines were used. 4×105 Capan1 and 2×105 BxPC3 and MiaPaCa2 cells were seeded in 75-cm2 flasks with 15 mL RPMI (Invitrogen) with added 0.1% gentamycin (Invitrogen) and 1% FCS (Invitrogen) and kept in continuous culture at 37°C in a humidified atmosphere (5% CO2) for four days. The culture media (conditioned media) were then collected, adjusted to 10% FCS, and used for the experiments with peripheral blood mononuclear cells (PBMC) within two hours after collection.
Human PBMC were isolated from a total of 40 blood donors’ buffy coats by differential density gradient centrifugation (Histopaque®-1077, Sigma-Aldrich, Milano, Italy). In the first series of experiments, PBMC from 17 donors were split into two or more fractions and cultured for four days (6×106 cells in each well of a 6 well culture plate) in complete control medium (RPMI, 10% FCS), or in pancreatic cancer cell conditioned media. After collection, the cells were analyzed under flow cytometry. PBMC from 7 donors were split into two fractions and cultured for two days (6×106 cells in each well of a 6 well culture plate) in complete control medium (RPMI, 10% FCS) in the absence or presence of 10 nM S100A8/A9 complex (DBA Italia srl, Milano, Italy) and then analysed by flow cytometry. In the second series of experiments, PBMC from 7 blood donors were cultured for 4 days in control and Capan1 conditioned media. After collection 50×106 cells were incubated in the dark for 30 minutes with 15 µL CD33-FITC, 20 µL HLA-DR-PC5, 15 µL CD14-PC7, 15 µL CD45-ECD. Cells that were CD33+CD14−HLA-DR+ were sorted (BD FACSAria III
The statistical analysis of data was made using the non parametric Kruskal-Wallis test, Wilcoxon signed ranks test, multiple comparison between groups with adjusted p-value, Cox proportional hazard model, non parametric test for trend across ordered groups, the Chi-Square test, and Spearman’s correlation, SPSS 9.0 and Stata (Statacorp, Texas, USA) statistical softwares being employed. Patients with splenic non-neoplastic lesions (6 cases) and those with chronic pancreatitis (7 cases) were considered overall as the reference group.
Patients’ details are reported in
No differences were found between patients for circulating and splenic CD4+ (Kruskal-Wallis test: p = 0.906 and p = 0.378) and CD4+CD25+ T cells (p = 0.596 and p = 0.420) or splenic CD8+ T cells (p = 0.290). On the contrary circulating CD8+ T cells significantly varied between groups (Kruskal-Wallis test: p = 0.004)(
Ref. = reference group made of patients with chronic pancreatitis (open dots) and of patients with splenic non-neoplastic lesions; SCA = Serous cystadenoma; BPNs = Borderline pancreatic neoplasms; PDAC = Ductal adenocarcinoma; NETs = Neuroendocrine tumors; Other = Non-pancreatic tumors. Each dot represents one case, and each open square represents five cases. * = p<0.0001 with respect to Ref. and p<0.001 with respect to BPNs.
Immature circulating and splenic myeloid cells were gated on the basis of CD33 expression levels, excluding lymphocytes and mature granulocytes. Depending on CD14 and HLA-DR expression levels, CD33+ cells were classified in four subsets: CD14+HLA-DR+, CD14+HLA-DR−, CD14−HLA-DR+ and CD14−HLA-DR−. Only CD14−HLA-DR+ circulating dendritic cells were correlated with disease diagnosis (Kruskal-Wallis Test: p = 0.01), lower levels being found in PDAC than in NETs (p = 0.003, adjusted p value for significance = 0.004). In spleen these cells tended to be higher in PDAC, NETs and borderline pancreatic tumors than in the reference cases (p = 0.077). The ratio between splenic and circulating CD14−HLA-DR+ was slightly higher in borderline tumors, but significantly higher in PDAC cases than in reference (Test for trend across ordered groups: p = 0.028) (
Ref. = reference group made of patients with chronic pancreatitis and of patients with splenic non-neoplastic lesions; BPNs = Borderline pancreatic neoplasms; PDAC = Ductal adenocarcinoma; NETs = Neuroendocrine tumors. Boxes represent interquartile ranges with medians; bars represents minimum and maximum values.
The cellular complexity that CD33+ cells presented at further evaluation was taken into account for a complete analysis of results. Three sets with low, intermediate and high complexity found in the whole CD33+ blood and splenic cell population are shown in
Panel A (upper left): a typical example of gating of low, intermediate (Int.) and high complexity sets among CD33+ cells in flow cytometry. Panel B (upper right): low, intermediate and high complexity CD33+ cells were analysed on the basis of CD14 and HLA-DR expression. A typical example is shown in this panel. Panel C (lower left): Blood low complexity CD33+CD14−HLA-DR+ cells. Panel D (lower right): blood low complexity CD33+CD14−HLA-DR− cells. Ref. = reference group made of patients with chronic pancreatitis (open dots) and of patients with splenic non-neoplastic lesions; BPNs = Borderline pancreatic neoplasms; PDAC = Ductal adenocarcinoma; NETs = Neuroendocrine tumors. * = p<0.004 (adjusted p-value for significance) with respect to Reference.
None of the four circulating immature myeloid cells subsets with intermediate or high complexity correlated with disease diagnosis (p>0.05 for all eight subsets). Among low complexity circulating immature myeloid cells, CD14−HLA-DR+ were significantly reduced (Kruskal-Wallis test: p = 0.026;
PDAC patients, subdivided based on the presence (n = 24) or absence (n = 28) of vascular invasion, had a median post-operative follow-up of 12.5 (range 1–23) months during which 18 patients died of disease-related causes.
Vascular invasion was significantly associated with a worse prognosis, as reported in
HR | 95% CI | p = | |
|
1.01 | 0.94–1.09 | 0.649 |
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0.96 | 0.31–3.03 | 0.953 |
|
6.04 | 1.62–22.54 | 0.007 |
HR = Hazard ratio; CI = confidence interval.
The studied circulating and splenic CD4+, CD8+ and CD4+CD25+ lymphocyte subsets were not correlated with the presence or absence of vascular invasion (p>0.05 for all subsets) and nor were they predictive of overall survival after surgery (Cox proportional hazard model corrected for age and sex: p>0.05 for all subsets).
No (n = 16) | Yes (n = 11) | |||
Median (IQR) | Median (IQR) | p-value | ||
|
|
84.2 (76.5–88.1) | 74.8 (71.4–83.4) | 0.008 |
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1.5 (0.4–3.9) | 7.7 (2.1–21.9) | 0.022 | |
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8.5 (6.2–12.5) | 10.4 (6.1–13.7) | 0.961 | |
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4.6 (3.2–8.3) | 4.3 (3.3–6.2) | 0.693 | |
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||
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27.7 (18.2–48.7) | 35.9 (31.2–52.9) | 0.223 |
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0.0 (0.0–0.4) | 0.6 (0.2–2.2) | 0.028 | |
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66.8 (47.4–69.9) | 57.2 (41.4–58.8) | 0.223 | |
|
5.9 (2.2–10.3) | 6.2 (3.3–10.3) | 0.935 |
Median values, interquartile range (IQR) and a statistical analysis (Kruskal-Wallis test) are reported.
In a series of 30 splenic samples (5 reference, 3 borderline tumors, 15 PDAC, 7 NETs), we studied the membranal expression of the two inhibitory co-stimulatory molecules PDL1 and CTLA4. PDL1 was highly expressed by immature CD14−HLA-DR+ cells in PDAC with respect to reference patients (p = 0.007, adjusted p value for significance = 0.008)(
Ref. = reference group made of patients with serous cystadenoma (open dots) and of patients with splenic non-neoplastic lesions; PaCa = Ductal adenocarcinoma; NETs = Neuroendocrine tumors. Kruskal-Wallis test: p = 0.046.
The fold increase of S100A8 and S100A9 mRNA, analysed in a series of 9 PDAC splenic samples, varied from 0 to 22 for S100A8 (median: 2.2, interquartile range: 0.6–5.7) and from 0 to 11.3 for S100A9 (median: 2.1, interquartile range: 0.5–3.4). S100A8 and S100A9 mRNA expression levels were correlated each other (Spearman’s r = 0.946, p<0.0001). Both S100A8 and S100A9 were correlated with the percentage of CD14+HLA-DR+PDL+ immature myeloid splenic cells (r = 0.819, p<0.001 for both), but not with the other studied lymphocyte and immature myeloid cell subsets.
PBMC from 17 healthy donors were analysed by flow cytometry to identify lymphocyte and immature myeloid cell subsets after they have been cultured for 4 days in control, BxPC3, Capan1 or MiaPaCa2 conditioned media.
Control (n = 17) | BxPC3 (n = 6) | Capan1 (n = 11) | MiaPaCa2 (n = 6) | |
Median (IQR) | Median (IQR) | Median (IQR) | Median (IQR) | |
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50 (44–57) | 58 (39–61) | 50 (41–51) | 56 (41–61) |
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0.674 | 0.386 | 0.917 | |
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23 (15–27) | 19 (15–31) | 22 (16–27) | 19 (15–32) |
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0.715 | 0.625 | 0.917 | |
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10 (9–11) | 9 (8–11) | 12 (10–13) | 9 (8–11) |
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0.269 | 0.014* | 0.599 | |
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71 (67–86) | 59 (36–74) | 81 (76–88) | 59 (40–77) |
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0.075 | 0.062 | 0.463 | |
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0.0 (0.0–0.4) | 0.2 (0.0–0.3) | 0.1 (0.0–0.4) | 0.0 (0.0–0.3) |
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0.465 | 0.391 | 0.715 | |
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16.5 (8–21) | 26.9 (15–47) | 10.8 (10–14) | 29.7 (12–46) |
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0.116 | 0.033* | 0.249 | |
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9.3 (3–13) | 14.3 (12–16) | 6.7 (1–11) | 9.7 (8–17) |
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0.028* | 0.285 | 1.00 |
A total of 17 healthy PBMC were analysed by flow cytometry after they have been cultured for 4 days in control medium or pancreatic cancer cell conditioned media. PBMC from 11 donors were cultured in Capan1 conditioned and in their respective control media, while PBMC from 6 donors were cultured in BxPC3 and MiaPaCa2 conditioned media and in their respective control media. The median and interquartile ranges (IQR) of the percentage of lymphocyte and CD33+ immature myeloid cell subsets are shown. The statistical analysis of data (Wilcoxon signed rank test) was made by pairing any conditioned media result with its own control. Asterisks highlight statistical significance.
CD14−HLA-DR+ dendritic cells from control and Capan1 conditioned PBMC obtained from a separate series of 8 healthy donors, were FACS sorted and co-cultured with allogenic T cells to assess their suppressive function. The proliferation of allogenic T cells co-cultured with control or Capan1 conditioned CD14−HLA-DR+ cells did not significantly differ when CD14−HLA-DR+/T cell ratio was at 1∶40 (Wilcoxon singed ranks test: p = 0.81) nor when it was 1∶20 (p = 0.54).
We then evaluated the percentage of PDL1 and CTLA4 positive cells among myeloid cell subsets in control and pancreatic cancer cell conditioned PBMC and results are shown in
Healthy PBMC were analysed by flow cytometry after they have been cultured for 2 days in the absence (Control) or presence of 10 nM S100A8/A9 heterocomplex. Immature myeloid cells were gated on the basis of CD33 expression. Panel A: percentage variations of CD14+HLA-DR− MDSCs; panel B: percentage variations of CTLA4 among CD14+HLA-DR− MDSCs; panel C: percentage variations of CD14−HLA-DR+ dendritic cells; panel D: percentage variations of PDL1 among CD14−HLA-DR+ dendritic cells.
Control (n = 14) | BxPC3 (n = 6) | Capan1 (n = 8) | MiaPaCa2 (n = 6) | |
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56 (37–73) | 61 (23–82) | 76 (73–92) | 50 (21–76) |
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0.249 | 0.036* | 0.686 | |
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31 (22–36) | 26 (10–50) | 46 (39–53) | 33 (28–45) |
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0.753 | 0.017* | 0.173 | |
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2.7 (0.4–4.8) | 3.3 (0.3–6.3) | 3.8 (1.3–10.6) | 1.7 (1.3–5.8) |
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0.753 | 0.108 | 0.345 | |
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16 (12–23) | 28 (9–54) | 6 (1–8) | 15 (10–20) |
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0.345 | 0.028* | 0.500 | |
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13 (4–21) | 4 (2–16) | 4 (1–7) | 10 (2–57) |
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0.116 | 0.043* | 0.917 | |
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12.3 (10.0–13.2) | 5.7 (0.5–10.7) | 4.3 (2.9–5.8) | 8.3 (2.9–19.5) |
|
0.028* | 0.046* | 0.753 |
Healthy PBMC were analysed by flow cytometry after they have been cultured for 4 days in control medium or in pancreatic cancer cell conditioned media. PBMC from 8 donors were cultured in Capan1 conditioned and in their respective control media, while PBMC from 6 donors were cultured in BxPC3 and MiaPaCa2 conditioned media and in their respective control media. In the series of 8 PBMC donors used for Capan1 experiments, CTLA4 data was available for a subset of 6 donors. Median and interquartile ranges (in brackets) of the percentage of CD33+ immature myeloid cells expressing PDL1 or CTLA4 are shown. Only few events among the CD33+CD14+HLA-DR− cell population were obtained (see
FACS sorted CD33+CD14−HLA-DR+PDL1+ cells did not significantly modify allogenic T cells proliferation with respect to CD33+CD14−HLA-DR+PDL1− cells (Wilcoxon singed ranks test: p = 0.11), despite they had a slight stimulatory effect (
Panel A: CD33+CD14−HLA-DR+PDL1+ and CD33+CD14−HLA-DR+PDL1− cells were FACS sorted and cocultured with allogenic T cells and proliferation was evaluated by (3H)-thymidine incorporation. Assay was performed in triplicate; data are mean ± SE of 4 independent experiments. Panel B: CTLA4+ and CTLA4− dendritic cells were FACS sorted and cocultured with allogenic T cells and proliferation was evaluated by (3H)-thymidine incorporation. Assay was performed in triplicate; data are mean ± SE of 3–4 independent experiments.
A dysregulation of myeloid and of T cells is now considered an emerging hallmark of cancer
Immature myeloid cells were selected by gating in the first line CD33 positive cells; four different subclasses were then defined on the basis of their high or low expression of the monocytic marker CD14, and of HLA-DR, a marker of mature dendritic cells
While the immature myeloid lineage marker CD33 is often taken into consideration for defining MDSCs
Among immature myeloid cells, those CD14+HLA-DR− are also considered MDSCs
Among the incoming cancer immunotherapy strategies, antibody blockade of the inhibitory molecules CTLA4 and PDL1 appears a promising approach
We then evaluated whether variations in PDL1 and CTLA4 expression are associated with an immunesuppressive phenotype. PDL1 expression characterized an overall stimulatory, not inhibitory, phenotype. To study whether CTLA4 is involved in immune suppression, experiments were performed using allogenic T cells co-cultured with dendritic cells. Dendritic cells were chosen for the experiments to obtain representative numbers of cellular events with positive or negative CTLA4 expression but also because dendritic cells are obtained by stimulating PBMC with GM-CSF, a cytokine recently demonstrated to be highly expressed by pancreatic cancer and necessary and sufficient for
In conclusion, in PDAC clinical setting an overall pattern of immune suppression prevails, characterized by reduced levels of circulating CD8+ T cells and dendritic cells and by increased circulating and splenic levels of MDSCs. A reduced expression of CTLA4 among splenic MDSCs was observed in PDAC patients and was demonstrated to be consequent to the direct exposure of myeloid cell to pancreatic cancer derived products and in particular to the S100A8/A9 complex. A reduced CTLA4 expression was also shown to be a feature of an immune suppressive phenotype and this suggests caution in the use of anti-CTLA4 therapies.