STAT3 mutation impacts biological and clinical features of T-LGL leukemia

STAT3 mutations have been described in 30-40% of T-large granular lymphocyte (T-LGL) leukemia patients, leading to STAT3 pathway activation. Considering the heterogeneity of the disease and the several immunophenotypes that LGL clone may express, the aim of this work was to evaluate whether STAT3 mutations might be associated with a distinctive LGL immunophenotype and/or might be indicative for specific clinical features.Our series of cases included a pilot cohort of 101 T-LGL leukemia patients (68 CD8+/CD4- and 33 CD4+/CD8\ub1) from Padua Hematology Unit (Italy) and a validation cohort of additional 20 patients from Rennes Hematology Unit (France).Our results indicate that i) CD8+ T-LGL leukemia patients with CD16+/CD56- immunophenotype identify a subset of patients characterized by the presence of STAT3 mutations and neutropenia, ii) CD4+/CD8\ub1 T-LGL leukemia are devoid of STAT3 mutations but characterized by STAT5b mutations, and iii) a correlation exists between STAT3 activation and presence of Fas ligand, this molecule resulting highly expressed in CD8+/CD16+/CD56- patients. Experiments with stimulation and inhibition of STAT3 phosphorylation confirmed this relationship. In conclusion, our data show that T-LGL leukemia with specific molecular and phenotypic patterns is associated with discrete clinical features contributing to get insights into molecular bases accounting for the development of Fas ligand-mediated neutropenia

Pancreatic Cancer (PaCa)-Derived Soluble Mediators Induce Dendritic Cells (DC) to Acquire an Immunosuppressive Phenotype by Downregulating CTLA4

Context An altered function of lymphocytes, DC and immature myeloid cells appears to be an hallmark of tumor-mediated immune suppression and the two inhibitory co-stimulatory receptors PDL-1 and CTLA4 might have a role in this context. Objective The aim of the present in vitro study was to assess whether PaCa cells cross-talk with normal mononuclear circulating cells (PBMC) causing them to acquire an immune\uadsuppressive phenotype and to evaluate whether PDL1 and CTLA4 are involved. Methods PBMC from blood donors were cultured for 4 days in control (CTL) and in the PaCa cancer cell line Capan1 conditioned media (CM). Lymphocytes subsets (CD4+, CD8+, CD4+CD25+) and CD33+ immature myeloid cells subsets (CD14+/-; HLA-DR+/-) expressing or not PDL1 and/or CTLA4 were analyzed by flow cytometry. To assess immunosuppressive function, myeloid cells were FACS sorted and co-cultured with allogenic total T lymphocytes in 1:20 and 1:40 ratios. Total T lymphocytes proliferation was determined by 3H-thymidine uptake. Results Capan1 CM caused an expansion of CD4+CD25+ (P=0.01) and a reduction of CD33+CD14-HLA-DR+ (P=0.03) cells. In this latter cellular subset, CM caused also an increase of PDL1 (P=0.046) and a decrease of CTLA4 (P=0.05) positive cells. FACS sorted CTL and CM CD33+CD14-HLA-DR+ cells did not significantly affect the proliferation of allogenic total T lymphocytes both at 1:20 (P=0.54) or at 1:40 ratios (P=0.81). The CD33+CD14-HLA-DR+ PDL-1+ cells did not significantly modify allogenic T cells proliferation with respect to PDL- cells (P=0.11), while those cells which were CTLA4 negative caused a significant inhibition of T cell proliferation in comparison of CTLA4 positive cells (P=0.008). Conclusions PaCa-derived soluble factors induce the expansion of the inhibitory lymphocyte subset CD4+CD25+ and a reduction of the immature CD33+CD14-DR+ dendritic cells. The tumor associated reduced expression of the inhibitory molecule CTLA4 in this cell population was demonstrated to characterize an immunosuppressive phenotype and this study suggests to take care in the use of anti-CTLA4 therapies

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

<p>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.</p

Baseline patients’ characteristics.

<p>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;</p>§<p>Other tumors included 3 papillary, 3 duodenal and 3 stromal tumors.</p>*<p>2/7 patients underwent middle pancreatectomies.</p

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

<p> 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.</p

CTLA4 negative dendritic cells suppress T cell proliferation.

<p>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.</p

Cox regression analysis of vascular invasion corrected for age and gender for survival in PDAC patients.

<p>HR = Hazard ratio; CI = confidence interval.</p

Pancreatic cancer cell conditioned media effects on lymphocyte and immature myeloid cell subsets.

<p>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.</p