Florian Cabillic

Soluble HLA-G molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic cells.

HLA‐G molecules are known to exert immunosuppressive action on DC maturation and on NK cells, and can in consequence inhibit respectively T cell responses and NK cytolysis. In this study, we show that monocyte‐derived DC, differentiated in the presence of GM‐CSF and IL‐4, are sensitive to soluble (s) HLA‐G molecules during LPS/IFN‐γ maturation as demonstrated by the decrease of CD80 and HLA‐DR expressions and IL‐12 secretion. Moreover, DC pretreated with sHLA‐G were found to activate NK/DC crosstalk less than non‐treated DC. Early activation of NK cells co‐cultured with autologous DC was diminished as assessed by CD69 expression. The IFN‐γ production was impaired whereas a slight inhibition of the NK cell cytotoxicity against Daudi cell line was observed. Since sHLA‐G is expressed in grafts or sites of tumour proliferation, its indirect action on NK cells via DC could constitute a pathway of early inhibition for both innate and specific immune responses.

DNAX accessory molecule-1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vγ9Vδ2 T cells

Human Vγ9Vδ2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway‐derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate. They display a strong cytotoxic activity against several tumor types, including hepatocellular carcinoma (HCC). Little is known about the mechanisms underlying Vγ9Vδ2 T‐cell recognition of tumor cells, but there is strong evidence that activating NK receptors play a role in γδ T‐cell cytotoxicity. In this study, we showed that the two NK receptors DNAX accessory molecule‐1 (DNAM‐1) and CD96 were expressed by Vγ9Vδ2 T cells. The ligands Nectin‐like‐5 specific of both DNAM‐1 and CD96, and also Nectin‐2, an additional ligand of DNAM‐1, were present on all HCC cell lines analyzed. Furthermore, we demonstrated by mAb‐mediated masking experiments that cytotoxicity against HCC cells as well as IFN‐γ production in γδ T cells were dependent on DNAM‐1. Our experiments indicated that Nectin‐like‐5 but not Nectin‐2 was involved in DNAM‐1‐dependent γδ T‐cell functions. We did not reveal a role for CD96 in the killing of HCC cells. Finally, we showed by combined mAb‐mediated blockade that DNAM‐1 and NKG2D could cooperate in the cell lysis of HCC.

Targeting RET in Patients With RET-Rearranged Lung Cancers: Results From the Global, Multicenter RET Registry

Purpose In addition to prospective trials for non-small-cell lung cancers (NSCLCs) that are driven by less common genomic alterations, registries provide complementary information on patient response to targeted therapies. Here, we present the results of an international registry of patients with RET-rearranged NSCLCs, providing the largest data set, to our knowledge, on outcomes of RET-directed therapy thus far. Methods A global, multicenter network of thoracic oncologists identified patients with pathologically confirmed NSCLC that harbored a RET rearrangement. Molecular profiling was performed locally by reverse transcriptase polymerase chain reaction, fluorescence in situ hybridization, or next-generation sequencing. Anonymized data-clinical, pathologic, and molecular features-were collected centrally and analyzed by an independent statistician. Best response to RET tyrosine kinase inhibition administered outside of a clinical trial was determined by RECIST v1.1. Results By April 2016, 165 patients with RET-rearranged NSCLC from 29 centers across Europe, Asia, and the United States were accrued. Median age was 61 years (range, 29 to 89 years). The majority of patients were never smokers (63%) with lung adenocarcinomas (98%) and advanced disease (91%). The most frequent rearrangement was KIF5B-RET (72%). Of those patients, 53 received one or more RET tyrosine kinase inhibitors in sequence: cabozantinib (21 patients), vandetanib (11 patients), sunitinib (10 patients), sorafenib (two patients), alectinib (two patients), lenvatinib (two patients), nintedanib (two patients), ponatinib (two patients), and regorafenib (one patient). The rate of any complete or partial response to cabozantinib, vandetanib, and sunitinib was 37%, 18%, and 22%, respectively. Further responses were observed with lenvantinib and nintedanib. Median progression-free survival was 2.3 months (95% CI, 1.6 to 5.0 months), and median overall survival was 6.8 months (95% CI, 3.9 to 14.3 months). Conclusion Available multikinase inhibitors had limited activity in patients with RET-rearranged NSCLC in this retrospective study. Further investigation of the biology of RET-rearranged lung cancers and identification of new targeted therapeutics will be required to improve outcomes for these patients.

Aminobisphosphonate-pretreated dendritic cells trigger successful Vγ9Vδ2 T cell amplification for immunotherapy in advanced cancer patients

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vγ9Vδ2 T lymphocytes represents an attractive strategy. Indeed, Vγ9Vδ2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vγ9Vδ2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vγ9Vδ2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vγ9Vδ2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vγ9Vδ2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vγ9Vδ2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vγ9Vδ2 T cells as measured by IFN-γ production. Moreover, we demonstrated that the cytotoxic level of Vγ9Vδ2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vγ9Vδ2 T cells leads eligible for Vγ9Vδ2 T cell adoptive immunotherapy the HCC and mCRC patients.

Daily regulation of serum and urinary hepcidin is not influenced by submaximal cycling exercise in humans with normal iron metabolism

Hepcidin and hemojuvelin (HJV) are two critical regulators of iron metabolism as indicated by the development of major iron overload associated to mutations in hepcidin and HJV genes. Hepcidin and HJV are highly expressed in liver and muscles, respectively. Intensive muscular exercise has been reported to modify serum iron parameters and to increase hepcidinuria. The present study aimed at evaluating the potential impact of low intensity muscle exercise on iron metabolism and on hepcidin, its key regulator. Fourteen normal volunteers underwent submaximal cycling-based exercise in a crossover design and various iron parameters, including serum and urinary hepcidin, were serially studied. The results demonstrated that submaximal ergocycle endurance exercise did not modulate hepcidin. This study also indicated that hepcidinuria did not show any daily variation whereas serum hepcidin did. The findings, by demonstrating that hepcidin concentrations are not influenced by submaximal cycling exercise, may have implications for hepcidin sampling in medical practice.

HLA-A*0201-restricted CEA-derived Peptide CAP1 Is Not a Suitable Target for T-cell-based Immunotherapy

Carcinoembryonic antigen (CEA) is a potential target for antigen-specific immunotherapy, as it is frequently overexpressed in human carcinomas. Moreover, an epitope derived from CEA, designated CAP1 (YLSGANLNL), has been proposed as naturally processed and presented by tumors in the human leukocyte antigen (HLA)-A*0201 context. Our aim was to fully characterize and assess the clinical relevance of the HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response against CEA. Stable and potent artificial antigen presenting cells (AAPCs) were used to evaluate T-cell response against CEA. These cells efficiently activate CTLs against tumor-derived epitopes after transduction with the antigenic peptides or full-length proteins. We found that AAPCs genetically modified to express CAP1, the agonist peptide CAP1-6D, or the whole CEA protein were not able to activate CAP1-specific CTLs from HLA-A*0201+ healthy donors or patients with colorectal carcinoma, even after multiple stimulations. In addition, we showed that a CAP1-specific T-cell clone, obtained after multiple stimulations of T cells of a HLA-A*0201+ healthy donor in vitro with autologous antigen presenting cells, recognized CEA− HLA-A*0201+ tumors transduced with a minigene encoding CAP1 but failed to react against HLA-A*0201+ tumor cells expressing CEA. Finally, AAPCs expressing the whole CEA protein did not induce any specific CTL response against CEA+ HLA-A*0201+ tumor cells highlighting the potential difficulty of mounting an efficacious T-cell response against this autoantigen. Altogether, our data indicate that CAP1 is not efficiently processed and presented by CEA+ tumor cells, and therefore, is not an appropriate target for T-cell-based immunotherapy.

Comparison of the immunomodulatory effects of L. donovani and L. major excreted-secreted antigens, particulate and soluble extracts and viable parasites on human dendritic cells

In an experimental model of human monocyte-derived dendritic cells (DCs), the immunophenotype of mature DCs infected with Leishmania donovani and Leishmania major showed a weak decrease in the cell surface expression of CD40, CD86, HLA-DR and DC-SIGN compared with uninfected control DCs. This immunomodulatory effect was more pronounced after stimulation with excreted-secreted antigens (ESA) of both species but absent after stimulation with particulate and soluble extracts. Infection with viable promastigotes, as well as stimulation with ESA from L. donovani and L. major, decreased IL-10 and IL-12p70 secretion. To our knowledge, this is the first direct demonstration that ESA from Leishmania promastigotes can stimulate DCs in the same manner as viable promastigotes.

Cytoplasmic PAR-3 protein expression is associated with adverse prognostic factors in clear cell renal cell carcinoma and independently impacts survival.

Clear cell renal cell carcinomas (ccRCCs) represent 70% of renal cancers, and several clinical and histolopathological factors are implicated in their prognosis. We recently demonstrated that the overexpression of PAR-3 protein encoded by the PARD3 gene could be implicated in renal oncogenesis. The object of this work was to study the association of intratumoral PAR-3 expression with known prognostic parameters and clinical outcome. In this aim, PAR-3 expression was assessed by immunohistochemistry in ccRCC tumors of 101 patients from 2003 to 2005. The immunostaining of PAR-3 was scored either as membranous (mPAR-3) or as both membranous and cytoplasmic (cPAR-3). Cytoplasmic PAR-3 was significantly associated with worse histopathological and clinical prognostic factors: Fuhrman grades 3 and 4, tumor necrosis, sarcomatoid component, adrenal invasion, renal and hilar fat invasion, eosinophilic component, a noninactivated VHL gene, higher tumor grade, lymph node involvement, metastasis, and worse clinical Eastern Cooperative Oncology Group and S classification scores. After multivariate analysis, 2 parameters were independently associated with cPAR-3: necrosis and eosinophilic components. In addition, cPAR-3 patients had shorter overall and progression-free survivals independently from strong prognostic validated factors like metastases. A cytoplasmic expression of PAR-3 is therefore implicated in worse clinical and pathological cancer features in ccRCC and could be useful to identify patients with high-risk tumors.

Overexpression of the polarity protein PAR‐3 in clear cell renal cell carcinoma is associated with poor prognosis

The partition‐defective 3 (PAR‐3) protein is implicated in the development and maintenance of cell polarity and is associated with proteins that mediate the changes in cytoskeleton organization required for cell polarity establishment. In this work, we used two original primary cell lines (R‐180 and R‐305) derived from clear cell Renal Cell Carcinoma (ccRCC) surgical specimens of a patient with unfavorable clinical course (R‐180 cells) and a patient with favorable prognosis (R‐305 cells) to identify genetic and molecular features that may explain the survival difference of the two patients. The cytogenetic analysis of these cell lines revealed that the PARD3 gene was amplified only in the R‐180 cell line that was derived from an aggressive ccRCC. PARD3 gene amplification was associated with overexpression of the encoded protein and altered cytoskeleton organization. Consistently, PARD3 knockdown in R‐180 cells restored the cytoskeleton organization and reduced cell migration in comparison to non‐transfected cells. Immunohistochemical analysis of ccRCC samples from a cohort of 96 patients with a follow‐up of 6 years revealed that PAR‐3 overexpression was correlated with poor survival. Our results suggest that PAR‐3 has a role in the clinical aggressiveness of ccRCC, possibly by promoting cell migration.

Histologic prognostic factors associated with chromosomal imbalances in a contemporary series of 89 clear cell renal cell carcinomas.

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. The aim of this study was to define specific chromosomal imbalances in ccRCC that could be related to clinical or histologic prognostic factors. Tumors and karyotypes of 89 patients who underwent nephrectomy for ccRCC were analyzed from April 2009 to July 2012. The mean number of chromosomal aberrations was significantly higher (7.8; P < .05) in Fuhrman grade 4 (F4) than in F3 (4) and F2 (3.4) cases. The results were similar, considering separately the mean number of chromosomal losses and gains. The F4 cases had a distinct pattern with more frequent losses of chromosomes 9, 13, 14, 18, 21, 22, and Y and gains of chromosome 20. Necrosis was associated with losses of chromosomes 7, 9, 18, and 22; sarcomatoid component, losses of chromosomes 7, 9, and 14 and gains of 20; and T stage, losses of chromosomes 18 and Y. After multivariate analysis, renal fat invasion, renal vein emboli, and microscopic vascular invasion were, respectively, associated with losses of chromosomes 13 and Y, loss of chromosome 13, and loss of chromosome 14 and gains of chromosomes 7 and 20. F4 was independently associated with losses of chromosomes 9 and Y; sarcomatoid component, loss of chromosome 9 and gain of 20; necrosis, loss of chromosome 18; and T stage, loss of chromosome Y. These chromosomal imbalances can be detected routinely by karyotype or fluorescence in situ hybridization analyses to stratify patients for risk of progression.