Pancreatic tumors and immature immunosuppressive myeloid cells in blood and spleen: role of inhibitory co-stimulatory molecules PDL1 and CTLA4. An in vivo and in vitro study

Abstract

Background: Blood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4.

Methodology and principal findings: 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 vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33(+)CD14(-)HLA-DR(-) were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33(+)CD14(+)HLA-DR(-) IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive.

Conclusion: 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.

Figure 1. Individual levels of CD8⁺ T cells in blood of the studied patients.

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.

Figure 2. Ratio between splenic and circulating CD33⁺CD14⁻HLA-DR⁺ immature myeloid cells.

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.

Figure 3. Immature myeloid cells in peripheral blood.

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.

Figure 4. Percentage of PDL-1 expression among splenic CD33⁺CD14⁻HLA-DR⁺ cells.

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.

Figure 5. S100A8/A9 induces PDL1 and inhibits CTLA4.

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.

Figure 6. CTLA4 negative dendritic cells suppress T cell proliferation.

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.