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Epithelial-to-mesenchymal transition (EMT) elicits mesenchymal stromal cell-like immune modulatory properties in cancer cells
S20
Poster Abstracts
46 EFFECT OF B CELL PURIFICATION AND CRYOPRESERVATION ON THE PRODUCTION OF HUMAN REGULATORY B CELLS FOR TRANSLATIONAL USE Y Wu1,2, A Pennati1, J Deng1, S Yuan1, M Garcia1, J Galipeau1,3 1 Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, United States, 2Department of Oncology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China, 3Department of Pediatrics, Winship Cancer Institute, Emory University, Atlanta, Georgia, United States Developing ex vivo regulatory B cells (Bregs) as a clinical intervention in autoimmune and inflammatory disorders is of translational interest. Mouse GIFT15, the chimeric protein between GM-CSF and IL-15, displayed immune suppressive function by converting naïve B cells into IL-10 producing Bregs (GIFT15-iBregs) in mice [PMID 19668193]. Hence, we hypothesize that human Bregs could be generated from peripheral blood mononuclear cells (PBMCs) with human GIFT15 in anticipation of testing the use of autologous Bregs as adoptive cell therapy. Our data showed that stimulating human PBMCs with GIFT15 converted naïve B cells to CD19+CD5+CD27+CD38+CD24 cells (termed human GIFT15-iBregs), which expressed substantial amount of IL-10 and suppressed both CD4+ and CD8+ T cell proliferation relative to naïve PBMCs (p<0.01). We tested whether B cell purification or cell cryopreservation prior to GIFT15 treatment would affect GIFT15-iBregs yield. Within 12 days’ expansion from 5.0106 naïve PBMCs, fresh unfractionated PBMCs generated 2.10.9106 GIFT15-iBregs, while purified B cells contracted to 0.140.02106 (p<0.05), and cryopreserved PBMCs produced 0.50.4106 (p<0.05). Interestingly, the post-thaw viability, GIFT15-iBregs yield, IL-10 expression and T cell suppressive function of cryopreserved GIFT15-stimulated PBMCs were not significantly different from fresh GIFT15-stimulated PBMCs. Further activation with GIFT15 after thawing maximized the function retention of cryopreserved GIFT15-iBregs. In conclusion, B cell purification and cryopreservation are feasible for the generation of functional GIFT15-iBregs but fresh unfractionated PBMCs as the starting material are optimal. Furthermore, cryopreservation of ex vivo generated GIFT15-iBregs can be successfully carried out without affecting their immunosuppressive properties and in vivo distribution. The development of human GIFT15-iBregs furthers the rationale of personalized Bregs adoptive therapy for immune disorders. 47 LOCAL ADOPTIVE IMMUNOTHERAPY WITH TH1 SPECIFIC AUTOLOGOUS LYMPHOCYTES MAY CORRECT THE CUTANEOUS HYPERTROPHIC SCAR GA Moviglia, MC Picon, J Huerta CIITT, Universidad Maimonides, Buenos Aires, Argentina Introduction: Dermal Hypertrophic Scar is one of the most often undesired side effects after a surgical closing or prosthesis implant of plastic surgery. The ethiopathogenesis of this process is related with a local Th2 cytokine environment. To overcome this process we have injected into the affected dermis tissue autologous Th1 normal dermis specific lymphocytes. Methods: Th1 dermis specific autologous Cells were selected from 200 ml of patient’s blood. Mononuclear cells of this blood sample were challenged during four days with dermis lysate in DMEM tissue culture media enriched with insulin, indomethacin and ranitidine. Elevated Interferon Gamma and Low IL6 production were considered signs of TH1 predominance in the cell suspension. Mononuclear cells were harvested, washed and injected into the dermis hypertrophic scar and the surrounded tissues. Previous the treatment and after it a skin biopsy was taken to perform a histologic study of the dermis changes. Results: Under compassionate uses, and after they signed an informed consent, five patients with chronic hypertrophic scar (more than 6 month of evolution) were treated with the above-described method. No one experience any undesired side effect. All of them experience changes of the visual and palpation exam showing diminishing or disappearance of the skin induration, as well as increasing of the dermis elasticity. The histologic study of these patients revealed diminishing of connective dense tissue which was replaced for soft connective tissue. These changes were associated with decreasing of collagen type 1 fiber density and increasing of reticular fibers presences. Conclusion: The present results suggest that local adoptive immunotherapy with Th1 specific autologous lymphocytes may correct the cutaneous hypertrophic scar. To prove these findings a phase 1-2 clinical trial should be conducted.
48 COMPARABILITY STUDY DURING MANUFACTURING PROCESS DEVELOPMENT OF OVASAVE, AN OVALBUMIN-SPECIFIC TYPE1 TREG IMMUNO-CELLULAR THERAPY OF REFRACTORY CROHN’S DISEASE N Belmonte, J Gertner, V Neveu, N Clerget, V Brun, E Pottier, M Forte, A Foussat TxCell S.A., Valbonne, France Introduction: Process development (P.Dev) of immuno-cellular therapies requires use of human cells. Due to difficult access to patient’s blood, P.Dev is performed with blood from healthy donors (HD). Final validation with patient’s cells is required to ensure process/product comparability and suitability of the process to for cells from inflamed donors. Methods: Ovasave is in clinical development for the treatment of Crohn’s disease (CD). Ovasave P.Dev in view of a Phase-IIb clinical study was performed with blood from HD. A clinical protocol submitted to the French regulatory agency and ethics committees, allowed obtaining CD patients’ blood for validation and comparability studies focused on purity, identity and product key attributes as well as in vitro safety. Results: Cellular non-Treg impurities of CD8 and Th17 type cells were absent in product from both HD and CD. Key product attributes such as ovalbuminspecificity and type 1 Treg cytokine profile were conserved with high IL-10 production and low levels of IFNg and IL-4. IL-10 production was indeed higher in CD. Expression of suppressive molecules such as CD39, GITR, Granzyme B and homing molecule expression were identical in HD and CD batches. Importantly, plasticity of Ovasave batches toward Th17 was absent in both CD and HD despite the published increased plasticity potential of Treg from CD patients. Time limited survival of cells composing Ovasave was comparable in both cases. Conclusion: Ovasave Phase-IIb production process developed with cells from HD is suitable Ovasave batches production from CD patient. Final validation with cells from CD allows obtaining batches with comparability in terms of purity, safety and expression of key effector suppressive molecules. 49 EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) ELICITS MESENCHYMAL STROMAL CELL-LIKE IMMUNE MODULATORY PROPERTIES IN CANCER CELLS M Ricciardi1, M Zanotto1, G Malpeli3, G Bassi1, O Perbellini1, M Chilosi2, F Bifari1, M Krampera1 1 Department of Medicine, Section of Hematology, University of Verona, Verona, VR, Italy, 2Department of Pathology and Diagnostics, Section of Pathological Anatomy, University of Verona, Verona, VR, Italy, 3Department of Surgery, University of Verona, Verona, VR, Italy Epithelial-to-Mesenchymal Transition (EMT) plays a central role in cancer progression and metastatic dissemination and may be induced by local inflammation. We asked whether the acquisition of mesenchymal phenotype by neoplastic epithelial cells is associated with the onset of mesenchymal stromal cell-like immune regulatory properties that may enhance tumor immune escape. Cell lines of lung adenocarcinoma (A549), breast cancer (MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B and NK cells before and after EMT induction by either the supernatant of mixed-lymphocyte reactions or inflammatory cytokines. EMT occurrence elicited multiple immune regulatory effects in cancer cells resulting in NK and T cell apoptosis, inhibition of lymphocyte proliferation and stimulation of regulatory T and B cells. Indoleamine 2,3-dioxygenase, but not Fas ligand pathway, was involved at least in part in these effects, as shown by the use of specific inhibitor. Thus, EMT induced by inflammatory stimuli confers to cancer cells some mesenchymal stromal cell-like immune modulatory properties, which could be a cue for cancer progression and metastatic dissemination by favoring immune escape.
50 ANTI-TUMOR EFFECT OF AUTOLOGOUS CYTOKINEINDUCED KILLER (CIK) CELLS IN PATIENTS WITH ADVANCED PRIMARY HEPOTIC CARCINOMA S Yang1, X Zhao1, J Huang1, H Li1, Y Yan2, W Dong2, L Huang1, Y Zhang1,2,3 1 Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China, 2Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China, 3School of Life Science, Zhengzhou University, Zhengzhou, Henan, China
Poster Abstracts
46 EFFECT OF B CELL PURIFICATION AND CRYOPRESERVATION ON THE PRODUCTION OF HUMAN REGULATORY B CELLS FOR TRANSLATIONAL USE Y Wu1,2, A Pennati1, J Deng1, S Yuan1, M Garcia1, J Galipeau1,3 1 Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, United States, 2Department of Oncology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China, 3Department of Pediatrics, Winship Cancer Institute, Emory University, Atlanta, Georgia, United States Developing ex vivo regulatory B cells (Bregs) as a clinical intervention in autoimmune and inflammatory disorders is of translational interest. Mouse GIFT15, the chimeric protein between GM-CSF and IL-15, displayed immune suppressive function by converting naïve B cells into IL-10 producing Bregs (GIFT15-iBregs) in mice [PMID 19668193]. Hence, we hypothesize that human Bregs could be generated from peripheral blood mononuclear cells (PBMCs) with human GIFT15 in anticipation of testing the use of autologous Bregs as adoptive cell therapy. Our data showed that stimulating human PBMCs with GIFT15 converted naïve B cells to CD19+CD5+CD27+CD38+CD24 cells (termed human GIFT15-iBregs), which expressed substantial amount of IL-10 and suppressed both CD4+ and CD8+ T cell proliferation relative to naïve PBMCs (p<0.01). We tested whether B cell purification or cell cryopreservation prior to GIFT15 treatment would affect GIFT15-iBregs yield. Within 12 days’ expansion from 5.0106 naïve PBMCs, fresh unfractionated PBMCs generated 2.10.9106 GIFT15-iBregs, while purified B cells contracted to 0.140.02106 (p<0.05), and cryopreserved PBMCs produced 0.50.4106 (p<0.05). Interestingly, the post-thaw viability, GIFT15-iBregs yield, IL-10 expression and T cell suppressive function of cryopreserved GIFT15-stimulated PBMCs were not significantly different from fresh GIFT15-stimulated PBMCs. Further activation with GIFT15 after thawing maximized the function retention of cryopreserved GIFT15-iBregs. In conclusion, B cell purification and cryopreservation are feasible for the generation of functional GIFT15-iBregs but fresh unfractionated PBMCs as the starting material are optimal. Furthermore, cryopreservation of ex vivo generated GIFT15-iBregs can be successfully carried out without affecting their immunosuppressive properties and in vivo distribution. The development of human GIFT15-iBregs furthers the rationale of personalized Bregs adoptive therapy for immune disorders. 47 LOCAL ADOPTIVE IMMUNOTHERAPY WITH TH1 SPECIFIC AUTOLOGOUS LYMPHOCYTES MAY CORRECT THE CUTANEOUS HYPERTROPHIC SCAR GA Moviglia, MC Picon, J Huerta CIITT, Universidad Maimonides, Buenos Aires, Argentina Introduction: Dermal Hypertrophic Scar is one of the most often undesired side effects after a surgical closing or prosthesis implant of plastic surgery. The ethiopathogenesis of this process is related with a local Th2 cytokine environment. To overcome this process we have injected into the affected dermis tissue autologous Th1 normal dermis specific lymphocytes. Methods: Th1 dermis specific autologous Cells were selected from 200 ml of patient’s blood. Mononuclear cells of this blood sample were challenged during four days with dermis lysate in DMEM tissue culture media enriched with insulin, indomethacin and ranitidine. Elevated Interferon Gamma and Low IL6 production were considered signs of TH1 predominance in the cell suspension. Mononuclear cells were harvested, washed and injected into the dermis hypertrophic scar and the surrounded tissues. Previous the treatment and after it a skin biopsy was taken to perform a histologic study of the dermis changes. Results: Under compassionate uses, and after they signed an informed consent, five patients with chronic hypertrophic scar (more than 6 month of evolution) were treated with the above-described method. No one experience any undesired side effect. All of them experience changes of the visual and palpation exam showing diminishing or disappearance of the skin induration, as well as increasing of the dermis elasticity. The histologic study of these patients revealed diminishing of connective dense tissue which was replaced for soft connective tissue. These changes were associated with decreasing of collagen type 1 fiber density and increasing of reticular fibers presences. Conclusion: The present results suggest that local adoptive immunotherapy with Th1 specific autologous lymphocytes may correct the cutaneous hypertrophic scar. To prove these findings a phase 1-2 clinical trial should be conducted.
48 COMPARABILITY STUDY DURING MANUFACTURING PROCESS DEVELOPMENT OF OVASAVE, AN OVALBUMIN-SPECIFIC TYPE1 TREG IMMUNO-CELLULAR THERAPY OF REFRACTORY CROHN’S DISEASE N Belmonte, J Gertner, V Neveu, N Clerget, V Brun, E Pottier, M Forte, A Foussat TxCell S.A., Valbonne, France Introduction: Process development (P.Dev) of immuno-cellular therapies requires use of human cells. Due to difficult access to patient’s blood, P.Dev is performed with blood from healthy donors (HD). Final validation with patient’s cells is required to ensure process/product comparability and suitability of the process to for cells from inflamed donors. Methods: Ovasave is in clinical development for the treatment of Crohn’s disease (CD). Ovasave P.Dev in view of a Phase-IIb clinical study was performed with blood from HD. A clinical protocol submitted to the French regulatory agency and ethics committees, allowed obtaining CD patients’ blood for validation and comparability studies focused on purity, identity and product key attributes as well as in vitro safety. Results: Cellular non-Treg impurities of CD8 and Th17 type cells were absent in product from both HD and CD. Key product attributes such as ovalbuminspecificity and type 1 Treg cytokine profile were conserved with high IL-10 production and low levels of IFNg and IL-4. IL-10 production was indeed higher in CD. Expression of suppressive molecules such as CD39, GITR, Granzyme B and homing molecule expression were identical in HD and CD batches. Importantly, plasticity of Ovasave batches toward Th17 was absent in both CD and HD despite the published increased plasticity potential of Treg from CD patients. Time limited survival of cells composing Ovasave was comparable in both cases. Conclusion: Ovasave Phase-IIb production process developed with cells from HD is suitable Ovasave batches production from CD patient. Final validation with cells from CD allows obtaining batches with comparability in terms of purity, safety and expression of key effector suppressive molecules. 49 EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) ELICITS MESENCHYMAL STROMAL CELL-LIKE IMMUNE MODULATORY PROPERTIES IN CANCER CELLS M Ricciardi1, M Zanotto1, G Malpeli3, G Bassi1, O Perbellini1, M Chilosi2, F Bifari1, M Krampera1 1 Department of Medicine, Section of Hematology, University of Verona, Verona, VR, Italy, 2Department of Pathology and Diagnostics, Section of Pathological Anatomy, University of Verona, Verona, VR, Italy, 3Department of Surgery, University of Verona, Verona, VR, Italy Epithelial-to-Mesenchymal Transition (EMT) plays a central role in cancer progression and metastatic dissemination and may be induced by local inflammation. We asked whether the acquisition of mesenchymal phenotype by neoplastic epithelial cells is associated with the onset of mesenchymal stromal cell-like immune regulatory properties that may enhance tumor immune escape. Cell lines of lung adenocarcinoma (A549), breast cancer (MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B and NK cells before and after EMT induction by either the supernatant of mixed-lymphocyte reactions or inflammatory cytokines. EMT occurrence elicited multiple immune regulatory effects in cancer cells resulting in NK and T cell apoptosis, inhibition of lymphocyte proliferation and stimulation of regulatory T and B cells. Indoleamine 2,3-dioxygenase, but not Fas ligand pathway, was involved at least in part in these effects, as shown by the use of specific inhibitor. Thus, EMT induced by inflammatory stimuli confers to cancer cells some mesenchymal stromal cell-like immune modulatory properties, which could be a cue for cancer progression and metastatic dissemination by favoring immune escape.
50 ANTI-TUMOR EFFECT OF AUTOLOGOUS CYTOKINEINDUCED KILLER (CIK) CELLS IN PATIENTS WITH ADVANCED PRIMARY HEPOTIC CARCINOMA S Yang1, X Zhao1, J Huang1, H Li1, Y Yan2, W Dong2, L Huang1, Y Zhang1,2,3 1 Biotherapy Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China, 2Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China, 3School of Life Science, Zhengzhou University, Zhengzhou, Henan, China