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Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death via Large Pore Formation.

Matsuoka S, Ishii Y, Nakao A, Abe M, Ohtsuji N, Momose S, Jin H, Arase H, Sugimoto K, Nakauchi Y, Masutani H, Maeda M, Yagita H, Komatsu N, Hino O - PLoS ONE (2016)

Bottom Line: We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line.This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation.This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo, 113-8421, Japan.

ABSTRACT
To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.

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Mechanism of mAb 4713-induced cell death involving the cytoskeleton.(A) Impact of chemical inhibitors on the cytotoxic activity of mAb 4713 against L428 and Raji cells. The inhibitors were added 1 or 2 h before the cytotoxicity assay. The percentage of dead cells was determined by trypan blue dye exclusion. **p = 0.01 versus none. ***p = 0.001 versus none. The agents were caspase inhibitors (z-VAD-FMK and z-Asp-DCB), PI-3 kinase inhibitors (wortmannin and LY294002), cytoskeletal inhibitors (cytochalasin D and latrunculin B), necroptosis inhibitor (Necrostatin-1), necrosis inhibitor (IM-54), a cathepsin inhibitor (Cath inib III), and reactive oxygen species scavenger (Tiron). (B) Limiting dilution experiments in which L428 cells were seeded (0.3 cells/well; 96 wells) with 3 μg/ml mAb 4713 or control IgG. After 2 h, the number of live cells per well was counted. **p = 0.01 versus isotype control. ***p = 0.001 versus control IgG. Each value represents the mean ± SD.
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pone.0150496.g004: Mechanism of mAb 4713-induced cell death involving the cytoskeleton.(A) Impact of chemical inhibitors on the cytotoxic activity of mAb 4713 against L428 and Raji cells. The inhibitors were added 1 or 2 h before the cytotoxicity assay. The percentage of dead cells was determined by trypan blue dye exclusion. **p = 0.01 versus none. ***p = 0.001 versus none. The agents were caspase inhibitors (z-VAD-FMK and z-Asp-DCB), PI-3 kinase inhibitors (wortmannin and LY294002), cytoskeletal inhibitors (cytochalasin D and latrunculin B), necroptosis inhibitor (Necrostatin-1), necrosis inhibitor (IM-54), a cathepsin inhibitor (Cath inib III), and reactive oxygen species scavenger (Tiron). (B) Limiting dilution experiments in which L428 cells were seeded (0.3 cells/well; 96 wells) with 3 μg/ml mAb 4713 or control IgG. After 2 h, the number of live cells per well was counted. **p = 0.01 versus isotype control. ***p = 0.001 versus control IgG. Each value represents the mean ± SD.

Mentions: The underlying mechanism of mAb 4713-induced cell death was investigated by testing the effects of several potential inhibitors on the cytotoxic activity of mAb 4713 against L428 HL cell line and Raji Burkitt lymphoma cell line. The caspase inhibitors, z-VAD-FMK [19] and z-Asp-DCB [20], and the PI-3 kinase inhibitors, wortmannin [21] and LY294002 [22], did not inhibit the cytotoxic activity of mAb 4713 (Fig 3A). To confirm the difference between mAb 4713 induced cell death from apoptosis, we performed several experiments. Western blot analysis supported the caspase-independent cell death mechanism. Treatment of Jurkat cells with cytochrome C showed bands of cleaved caspase-3, but treatment of L428 cells with mAb 4713 did not (S3A Fig). Similarly cleaved caspase-3 was not detected in L428 cells treated with mAb 4713 by flow cytometric analysis (S3B Fig). Furthermore, depolarization of mitochondrial membrane was not observed during mAb 4713-induced cell death (S4 Fig). The necroptosis inhibitor, Necrostatin-1 [23], and the necrosis inhibitor, IM-54 [24], also failed to inhibit the cytotoxic activity of mAb 4713 (Fig 4A). Consequently, mAb 4713-induced cell death is not apoptosis, necroptosis, nor necrosis. In contrast, both cytochalasin D, which depolymerizes cytoskeletal actin filaments to actin monomers [25], and latrunculin B, which reduces the monomeric actin pool available for polymerization [26], completely inhibited mAb 4713-induced cell death (Fig 4A). These data are reminiscent of previous findings regarding the cytolytic activity of anti-pan MHC class I mAb against lymphocytes [11, 12]. Therefore, cytolytic anti- pan MHC class I and class II antibodies may use the shared signaling events to induce cell death. We hypothesized that cell death was initiated by a disorganization of cytoskeletal actin filament systems induced by intensive cross-linking between anti-pan MHC mAbs and abundant cell surface MHC molecules because it was reported that Fab fragments of anti-pan MHC mAbs have no cytolytic activity, whereas cross-linking of Fab with anti-mouse Igs reconstituted cytotoxicity in T cells ([11] and our unpublished data].


Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death via Large Pore Formation.

Matsuoka S, Ishii Y, Nakao A, Abe M, Ohtsuji N, Momose S, Jin H, Arase H, Sugimoto K, Nakauchi Y, Masutani H, Maeda M, Yagita H, Komatsu N, Hino O - PLoS ONE (2016)

Mechanism of mAb 4713-induced cell death involving the cytoskeleton.(A) Impact of chemical inhibitors on the cytotoxic activity of mAb 4713 against L428 and Raji cells. The inhibitors were added 1 or 2 h before the cytotoxicity assay. The percentage of dead cells was determined by trypan blue dye exclusion. **p = 0.01 versus none. ***p = 0.001 versus none. The agents were caspase inhibitors (z-VAD-FMK and z-Asp-DCB), PI-3 kinase inhibitors (wortmannin and LY294002), cytoskeletal inhibitors (cytochalasin D and latrunculin B), necroptosis inhibitor (Necrostatin-1), necrosis inhibitor (IM-54), a cathepsin inhibitor (Cath inib III), and reactive oxygen species scavenger (Tiron). (B) Limiting dilution experiments in which L428 cells were seeded (0.3 cells/well; 96 wells) with 3 μg/ml mAb 4713 or control IgG. After 2 h, the number of live cells per well was counted. **p = 0.01 versus isotype control. ***p = 0.001 versus control IgG. Each value represents the mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814124&req=5

pone.0150496.g004: Mechanism of mAb 4713-induced cell death involving the cytoskeleton.(A) Impact of chemical inhibitors on the cytotoxic activity of mAb 4713 against L428 and Raji cells. The inhibitors were added 1 or 2 h before the cytotoxicity assay. The percentage of dead cells was determined by trypan blue dye exclusion. **p = 0.01 versus none. ***p = 0.001 versus none. The agents were caspase inhibitors (z-VAD-FMK and z-Asp-DCB), PI-3 kinase inhibitors (wortmannin and LY294002), cytoskeletal inhibitors (cytochalasin D and latrunculin B), necroptosis inhibitor (Necrostatin-1), necrosis inhibitor (IM-54), a cathepsin inhibitor (Cath inib III), and reactive oxygen species scavenger (Tiron). (B) Limiting dilution experiments in which L428 cells were seeded (0.3 cells/well; 96 wells) with 3 μg/ml mAb 4713 or control IgG. After 2 h, the number of live cells per well was counted. **p = 0.01 versus isotype control. ***p = 0.001 versus control IgG. Each value represents the mean ± SD.
Mentions: The underlying mechanism of mAb 4713-induced cell death was investigated by testing the effects of several potential inhibitors on the cytotoxic activity of mAb 4713 against L428 HL cell line and Raji Burkitt lymphoma cell line. The caspase inhibitors, z-VAD-FMK [19] and z-Asp-DCB [20], and the PI-3 kinase inhibitors, wortmannin [21] and LY294002 [22], did not inhibit the cytotoxic activity of mAb 4713 (Fig 3A). To confirm the difference between mAb 4713 induced cell death from apoptosis, we performed several experiments. Western blot analysis supported the caspase-independent cell death mechanism. Treatment of Jurkat cells with cytochrome C showed bands of cleaved caspase-3, but treatment of L428 cells with mAb 4713 did not (S3A Fig). Similarly cleaved caspase-3 was not detected in L428 cells treated with mAb 4713 by flow cytometric analysis (S3B Fig). Furthermore, depolarization of mitochondrial membrane was not observed during mAb 4713-induced cell death (S4 Fig). The necroptosis inhibitor, Necrostatin-1 [23], and the necrosis inhibitor, IM-54 [24], also failed to inhibit the cytotoxic activity of mAb 4713 (Fig 4A). Consequently, mAb 4713-induced cell death is not apoptosis, necroptosis, nor necrosis. In contrast, both cytochalasin D, which depolymerizes cytoskeletal actin filaments to actin monomers [25], and latrunculin B, which reduces the monomeric actin pool available for polymerization [26], completely inhibited mAb 4713-induced cell death (Fig 4A). These data are reminiscent of previous findings regarding the cytolytic activity of anti-pan MHC class I mAb against lymphocytes [11, 12]. Therefore, cytolytic anti- pan MHC class I and class II antibodies may use the shared signaling events to induce cell death. We hypothesized that cell death was initiated by a disorganization of cytoskeletal actin filament systems induced by intensive cross-linking between anti-pan MHC mAbs and abundant cell surface MHC molecules because it was reported that Fab fragments of anti-pan MHC mAbs have no cytolytic activity, whereas cross-linking of Fab with anti-mouse Igs reconstituted cytotoxicity in T cells ([11] and our unpublished data].

Bottom Line: We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line.This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation.This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo, 113-8421, Japan.

ABSTRACT
To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.

Show MeSH
Related in: MedlinePlus