Willy Berlier

Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction

Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3α/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up‐regulated by the proinflammatory cytokine interleukin‐1β through the nuclear factor‐κB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.

Innovative approach in Pompe disease therapy: Induction of immune tolerance by antigen-encapsulated red blood cells.

Pompe disease is a glycogen storage disease caused by acid α-glucosidase enzyme deficiency. Currently, the unique treatment is lifelong enzyme replacement therapy ERT with frequent intravenous administration of the recombinant analog alglucosidase-α (AGA), which ultimately generates a sustained humoral response resulting in treatment discontinuation. Our aim is to use the tolerogenic properties of antigen-encapsulated red blood cells (RBCs) to abolish the humoral response against AGA and to restore tolerance to replacement therapy. To demonstrate that our approach could prevent the AGA-induced immune response, mice were intravenously injected three times with AGA encapsulated into RBCs before being sensitized to AGA with several adjuvant molecules. Control animals received injections of free AGA instead of the encapsulated molecule. One-week after treatment with AGA-loaded RBCs, a strong decrease in specific humoral response was observed despite three stimulations with AGA and adjuvant molecules. Furthermore, this specific immunomodulation was maintained for at least two months without affecting the overall immune response. AGA-loaded RBCs represent a promising strategy to induce or restore tolerance in Pompe disease patients who develop hypersensitivity reactions following repeated AGA administrations.

Opposite immune reactivity of serum IgG and secretory IgA to conformational recombinant proteins mimicking V1/V2 domains of three different HIV type 1 subtypes depending on glycosylation.

The V1/V2 domain of the HIV-1 gp120 envelope protein has been shown to contribute to viral cell tropism during infection and also to viral recognition by neutralizing monoclonal antibodies. However, this domain has been poorly investigated. Carbohydrates have been demonstrated to dramatically influence immune reactivity of antisera to viral glycoprotein antigens. In this study, DNA sequences coding for V1/V2 domains from HIV-1 primary isolates of three subtypes (A, B, and C) were subcloned into a secretion vector and used to transfect CHO cells that are able to achieve the glycosylation of proteins. The structure of purified recombinant V1/V2 proteins was tested using two anti-V1/V2 monoclonal antibodies directed against either a linear or a conformational and glycosylation-dependent epitope (8.22.2 and 697-D). Serum or saliva of 14/82 seropositive patients with anti-V1/V2 reactivity demonstrated good recognition of the recombinant proteins. Deglycosylation of the recombinant proteins was found to increase the reactivity of the serum IgG to the clade A and C but not to clade B V1/V2 domain demonstrating that the recognition of glycosylation sites by serum IgG is clade dependent. When considering SIgA from parotid saliva, deglycosylation of all recombinant proteins tested decreased the reactivity, suggesting that glycosylation plays an important role in the recognition of V1/V2 domain target epitopes by this class of antibodies. In conclusion, these results suggest the influence of carbohydrate moieties on the specificity of the antibodies to the V1/V2 domain produced during HIV infection and the potential importance of viral glycans in vaccine responses after mucosal administration.

Construction and tropism characterisation of recombinant viruses exhibiting HIV-1 env gene from seminal strains.

Genetic differences between blood and mucosal-derived HIV-1 strains have been widely reported. As amplification of HIV-1 strains from mucosal samples including semen or saliva by co-culture has low sensitivity, we developed the construction of chimeric viruses expressing wild-type seminal HIV-1 envelope protein. Chimeric viruses were produced by co-transfection of a V1-V3 deleted pNL 43 vector and PCR fragments spanning the deleted region, amplified from HIV-1 RNA positive seminal plasma samples. After an initial testing of co-receptor usage by a tropism recombinant test, replication capacity and amplification of these recombinant viruses were assessed using PBMC. Four chimeric replicative strains, all using CXCR4 as coreceptor, were produced. The interaction between cell-free viral particles and reporter cell lines was assessed by confocal microscopy. These replicative chimeras exhibiting HIV-1 env from seminal strains represent useful tools for the in vitro study of the heterosexual transmission of HIV-1 and testing of microbicide activity.

Abstract 2356: Erythrocytes used as tumor antigen delivery system to target antigen-presenting cells embody an innovative approach for in situ cancer immunotherapy

Introduction: In current vaccine therapy, efficient delivery of tumor antigens (TA) to antigen-presenting cells (APC) represents a major step toward the development of strong TA-specific immune response including cytotoxic T lymphocyte (CTL) induction. Our innovative approach is to use the property of erythrocytes to be naturally phagocytosed by APC when senescent. Indeed, TA can be encapsulated into erythrocytes that will be used as carrier to specifically deliver the TA to APC, which will ensure their degradation and presentation to T cells. Materials and Methods: The proof of concept was established with Ovalbumin (OVA), tyrosinase-related protein 2 (TRP2 (melanoma TA) and prostate specific antigen (PSA, prostate TA). These TA were encapsulated into erythrocytes by a hypotonic lysis process. To stimulate the erythrophagocytosis by APC, the membranes of erythrocytes were coated with antibodies. Then, erythrocytes containing TA were intravenously injected to mice concomitantly with Poly(I:C) adjuvant. The CTL response was analyzed by IFNγ ELISPOT and in vivo lysis of TA-expressing target cells injected to immunized mice. Humoral response was assessed by the determination of TA-specific immunoglobulin titer in mouse serum. Results: All TA were efficiently encapsulated in erythrocytes in a dose-dependent manner. A strong T-cell response was induced after 2 injections of TA encapsulated in erythrocytes: 2μg of OVA and 20μg of TRP2 induced the in vivo lysis of 97% and 96% of TA-target cells, respectively. By comparison, injections of free antigen induced less than 5% of lysis. Furthermore, a significant number of TA-specific IFNγ-secreting cells was generated (1585 and 286 cells / 106 cells for TRP2 and PSA, respectively) compared to the free TA ( Conclusion: The use of erythrocytes as TA carrier to specifically deliver antigen to APC and induce efficient immune response against tumor can be a very promising strategy in cancer immunotherapy. Citation Format: Magali Cremel, Nathalie Guerin, Quitterie Barthe, Vanessa Bourgeaux, Willy Berlier, Francoise Horand, Yann Godfrin. Erythrocytes used as tumor antigen delivery system to target antigen-presenting cells embody an innovative approach for in situ cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2356.

Abstract 4812: Arginine deiminase loaded in erythrocytes: a promising formulation for L-arginine deprivation therapy in cancers

Based on the asparaginase paradigm, several arginine-catabolizing enzymes have been developed for the treatment of arginine-dependent cancers (Wheatley, 2004). The arginine deiminase (ADI) enzyme catalyzes the hydrolysis of arginine (Arg) (Sugimura, 1990). ADI purified from Mycoplasma has a short half-life (≈4h) in the circulation and was found to be highly immunogenic (Holstberg, 2002). In order to increase half-life and to limit immunogenicity, a pegylated form of the enzyme (ADI-PEG-20) was developed. Phase I/II clinical studies in hepatocarcinoma and in advanced melanoma concluded on its limited efficacy at the tested doses. Notably, reduction of the duration of Arg depletion was linked to the emergence of ADI-PEG-20 antibodies (Ascierto, 2006). Encapsulation of ADI into red blood cells (RBC) is a promising alternative to improve the half-life and reduce the immunogenicity of the protein. Argininosuccinate synthase (ASS1) is the key enzyme in arginine biosynthesis (Haines, 2011). ASS1 expression varies in tumors and ASS1 loss is associated with poor prognosis in different cancers (Qiu, 2015). All these data strengthen the importance of selecting ASS1-negative patients for Arg-depletion based enzymatic therapy. Using a scalable, standardized production process, we synthesized an ADI enzyme from an optimized M. arginini coding sequence. ADI was encapsulated into RBC by hypotonic dialysis (ERY-ADI) and PK-PD parameters were evaluated in CD1 mice, in comparison with free ADI. Sensitivity to ADI was assessed in vitro by measuring the cell viability of 3 cancer cell lines with different ASS1 expression levels. ASS1 expression was screened by immunohistochemistry (IHC) in a large panel of tissue microarrays (TMA) from 16 human cancer types. Administration of ERY-ADI (5.5IU/mL) reduced mouse plasmatic Arg level to 30% of control values and led to a maintained depletion for 5 days. The same dose of free ADI strongly depleted Arg ( All these results highlight that arginine depletion through ADI treatment is effective against ASS1-negative cancer cells. ERY-ADI represents an innovative product with an improved efficacy for sustained Arg depletion and suitable for the treatment of ASS1 deficient cancers. Citation Format: Fabien Gay, Karine Aguera, Karine Senechal, Julie Bes, Anne-Marie Chevrier, Fanny Gallix, Christine Guicher, Philip Lorenzi, Vanessa Bourgeaux, Willy Berlier, Francoise Horand, Yann Godfrin. Arginine deiminase loaded in erythrocytes: a promising formulation for L-arginine deprivation therapy in cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4812.

Abstract 5369: Low asparagine synthetase expression and in vitro sensitivity highlights L-asparaginase potential for the treatment of aggressive lymphomas

L-asparaginase (L-ASPA) is included in current chemotherapy regimen for the treatment of acute lymphoblastic leukemia (ALL). The sensitivity of ALL to L-ASPA has been linked to the incapacity of leukemic cells to produce their own asparagine, since they are deficient in a specific enzyme, asparagine synthetase (ASNS). Thus, ASNS-negative leukemic cells rely on plasmatic asparagine and can be starved to death by L-ASPA treatment. Several studies evidenced the potential of asparagine depletion to treat lymphomas. Indeed, L-ASPA is routinely administered to treat canine lymphomas and its adjunction in chemotherapy regimens significantly improves the outcome of patients with NK/T cell lymphoma. Some studies suggest its benefit for the treatment of B and T-cell non-Hodgkin lymphomas (NHL). In this study, we assessed the in vitro sensitivity to L-ASPA of 11 lymphoma cell lines, including diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL) and other NHL cell lines. We analyzed ASNS expression in tissue microarrays and biopsies from 226 cases of lymphomas, including 158 DLBCL, 15 PTCL, as well as other NHL and Hodgkin lymphomas. Sensitivity to L-ASPA (expressed as an IC50) was assessed by measuring the cell viability in the presence of various concentrations of L-ASPA. ASNS expression was assessed with a validated immunohistochemistry method attributing a score based on labeling intensity from 0 (no expression) to 3 (strong expression). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. All lymphoma cell lines were proved to be sensitive to L-ASPA. Their in vitro sensitivity usually exceeded well-known sensitive cell lines (MOLT-4 and HL-60) representative of acute leukemia. ASNS expression was null/low in 171/226 (76%) total cases of lymphomas. 118/158 (75%) DLBCL cases expressed no/low ASNS, whereas ASNS expression was low/null in 2/15 (13%) PTCL cases. Globally, B-cell NHL displayed a lower expression of ASNS than T-cell NHL, Hodgkin lymphoma and non tumoral lymph nodes. The in vitro sensitivity to L-ASPA of all lymphoma cell lines and the low/null expression of ASNS in the majority of the lymphoma tissues tested suggest that L-ASPA may be effective for the treatment of various lymphomas. As suggested by ASNS expression, L-ASPA treatment may be particularly effective in DLBCL and others B-cell NHL, whereas its efficacy in T-cell NHL is probably more limited. However, L-ASPA has only been used scarcely in the treatment of lymphomas despite promising clinical responses. Its well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis…) render its use particularly hazardous in older or frail patients. The development of a new formulation of L-ASPA with better safety profile must be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Anne-Marie Chevrier, Alexandra Traverse-Glehen, Yann Godfrin. Low asparagine synthetase expression and in vitro sensitivity highlights L-asparaginase potential for the treatment of aggressive lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2015-5369