The acetylcholine receptor (AChR) is the major autoantigen in the antibody-mediated disease myast... more The acetylcholine receptor (AChR) is the major autoantigen in the antibody-mediated disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). This study demonstrates that rats immunized with a recombinant fragment corresponding to the normally exposed extracellular region of the rat AChR alpha-subunit first develop antibodies to the injected extracellular portion only, but later develop antibodies to intracellular cytoplasmic epitopes of AChR. The presence of autoantibodies to intracellular epitopes seems to be correlated with development of clinical signs of disease. We propose that a similar process of epitope spreading may take place in the natural course of myasthenia.
We have previously demonstrated that the chemokine IFN-gamma inducible protein 10 (IP-10) and its... more We have previously demonstrated that the chemokine IFN-gamma inducible protein 10 (IP-10) and its receptor CXCR3, are overexpressed in myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG). We now studied the potential of modulating rat EAMG by interference in CXCR3/IP-10 signaling. Two different approaches were used: 1) blocking IP-10 by IP-10-specific antibodies and 2) inhibiting the CXCR3 chemokine receptor by a CXCR3 antagonist. Treatment by either of these reagents led to suppression of EAMG suggesting that inhibition of CXCR3/IP-10 signaling can be considered as a potential treatment modality for MG.
Protein databases were searched for microbial sequences that bear amino acid similarities with id... more Protein databases were searched for microbial sequences that bear amino acid similarities with identified T- or B-cell epitopes within the human alpha-subunit of acetylcholine receptor (AChR). One peptide, derived from Haemophilus influenzae, exhibits 50% homology to an identified T-cell epitope of AChR alpha-subunit. This peptide was shown to have a protective effect in experimental autoimmune myasthenia gravis (EAMG). Pretreatment of rats with the mimicry peptide attenuated the induction and progression of EAMG. These effects were accompanied by a reduced T-cell response to AChR, diminished IL-2, IL-12, IFN-gamma and IL-4 levels, as well as decreased humoral response to self-AChR.
Background: [corrected] Intravenous immunoglobulin administration has been beneficially used for ... more Background: [corrected] Intravenous immunoglobulin administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis, although its mode of action and active components have not yet been fully identified. Objectives: To isolate from IVIg a disease-specific fraction involved in the therapeutic activity in myasthenia and to identify its properties and function. Results: IVIg administration in experimental autoimmune MG results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of IVIg on columns of IgG from rats with EAMG or from MG patients resulted in depletion of the suppressive activity that IVIg has on rat EAMG. Moreover, the minute amounts of IgG fractions eluted from the EAMG or MG-specific columns retained the immunosuppressive activity of IVIg. Conclusions: Our study supports the notion that the therapeutic effect of IVIg is mediated by a minor disease-specific immunoglobulin fraction that is present in IVIg and is essential for its therapeutic activity.
Annals of the New York Academy of Sciences, Sep 1, 2007
Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of ... more Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis (MG). We have demonstrated that IVIG administration in experimental autoimmune MG (EAMG) results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of pooled human immunoglobulins (IVIGs) on immobilized IgG, isolated from rats with EAMG, results in a complete depletion of the suppressive activity of the IVIG. Moreover, the eluate from this EAMG-specific antibody column retains the immunosuppressive activity of IVIG. This study supports the notion that the therapeutic effect of IVIGs is mediated by an antigen-specific anti-immunoglobulin (anti-idiotypic) activity that is essential for its suppressive activity.
Annals of the New York Academy of Sciences, Jun 1, 2008
Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of ... more Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis (MG). We have demonstrated that IVIG administration in experimental autoimmune MG (EAMG) results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of pooled human immunoglobulins (IVIG) on immobilized IgG, isolated from rats with EAMG or from MG patients, results in a depletion of the suppressive activity of the IVIG. Moreover, reconstitution of the activity‐depleted IVIG with the eluted minute IVIG fractions that had been adsorbed onto the EAMG‐ or MG‐specific columns recovers the depleted immunosuppressive activity. This study supports the notion that the therapeutic effect of IVIG is mediated by an antigen‐specific anti‐immunoglobulin (anti‐idiotypic) activity that is essential for its suppressive activity.
Intravenous immunoglobulin (IVIG) treatment is beneficially used in autoimmune disorders includin... more Intravenous immunoglobulin (IVIG) treatment is beneficially used in autoimmune disorders including myasthenia gravis (MG) although its mode of action and active components are still not fully identified. In an attempt to isolate from IVIG a disease-specific suppressive fraction, IVIG was passed on columns of IgG from rats with experimental autoimmune MG (EAMG) or from MG patients. These chromatographies resulted in depletion of the suppressive activity of IVIG on rat EAMG whereas the minute amounts of IgG fractions eluted from the EAMG- or MG-specific columns retained the immunosuppressive activity of IVIG. These results demonstrate that a minor disease-specific immunoglobulin fraction present in IVIG is essential for its suppressive activity.
Introduction: Immunotherapies that harness CTLs to eradicate solid tumors, melanoma in particular... more Introduction: Immunotherapies that harness CTLs to eradicate solid tumors, melanoma in particular, have re-emerged as potent clinical options in recent years. Improving the dispersion and cytotoxicity of intratumoral CTLs may further benefit patients. To gain insight into the regulatory processes that limit CTL activity in melanoma we investigated the influence of PD-1 signaling and Tregs on CTL behavior. Methodology: To mimic adoptive cell therapy we transferred mice bearing B16.OVA melanoma tumors with OT-I CTLs. To study Treg-mediated regulation, we used FOXP3-GFP-LuciDTR-4 host mice, in which 95% of the cells expressing Foxp3 could be depleted using diphtheria toxin. To study PD-1 mediated regulation we injected anti PD-1 to WT host mice. Tumor rejection was monitored for 2-5 weeks. Two-photon imaging of live mice was used to follow the behavior of CTLs and Tregs within tumors. Time-lapse confocal microscopy was used to follow the influence of PD-1 signaling on the behavior of CTLs and their interaction with B16-Ova cells. Results: Tumor rejection was closely regulated by Tregs as tumors were densely infiltrated by these cells and their depletion dramatically accelerated tumor rejection. Interactions between CTLs and Tregs seemed indirect, as they did not form conjugates. Following their transfer, antigen-specific OT-I CTLs, as well as polyclonal CTLs, entered murine tumors through selected ICAM-decorated intratumoral vessels. As CTLs accumulated, Treg numbers in the tumors dropped and CTLs could be observed conjugating tumor cells and killing them. Treatment with anti PD-1 increased CTL velocities and accelerated killing of B16.OVA cells in vitro, and was mirrored in vivo by enhanced tumor rejection. Whereas CTL migration and cytolysis was limited to oxygenated tumor areas within 50 µm of flowing blood vessels, Tregs exhibited higher velocities, were not affected by the proximity of blood vessels, and penetrated deeper into avascular areas. Conclusions: Our findings suggest that regulatory mechanisms influence CTL efficiency partly by affecting their migration. CTLs, more than Tregs, depend on oxygen for intratumoral locomotion and cytolysis. This disparity allows tumor cells to escape CTL lysis in deep hypoxic tumor niches where high Treg to CTL ratios favor their survival. Thus, differential motility and dependence on oxygen among CTLs and Tregs can dictate the efficiency of CTL-based therapies of melanoma. Citation Format: Tali Feferman, Natalia G. Zabolinsky, Yoav Manaster, Anat Hutzler, Guy Shakhar. Imaging regulatory mechanisms that limit intratumoral CTL function in a mouse melanoma model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B06.
Background: One of the obstacles to successful immunotherapy by CTLs is thought to be poor vascul... more Background: One of the obstacles to successful immunotherapy by CTLs is thought to be poor vascularization of tumors that leads to sub-optimal tumor infiltration. In contrast, the effects of disrupted vascularization and the hypoxia it introduces on CTL migration and function within tumors have not been investigated. Methods: To study whether tumor vascularity can shape anti-tumor CTL responses, we combined immunohistochemistry of human melanoma samples with dynamic two-photon imaging within B16-OVA tumors in live mice. Results: In patients, CD8 T cells were twofold denser around peripheral vessels than around deep vessels, and fourfold denser than in avascular areas. This pattern was clearest in poorly-infiltrated tumors. Correspondingly, in mice, OT-I CTLs preferentially infiltrated B16-OVA tumors through peripheral ICAM1+ vessels. CTLs remained initially at their entry areas, crawling vigorously only within 50 µm of flowing blood vessels. Gradually, CTLs formed dense perivascular swarms, producing locally-high CTL-to-target ratios at their advancing front. These swarms advanced through the tumors, leading to patchy elimination of tumor cells. Target killing was antigen-specific, contact-dependent and perforin-mediated, and required several CTLs to engage one target cells to be effective. Several findings suggest that CTL confinement to perivascular areas is oxygen-dependent: occlusion of intratumoral blood vessels resulted in immediate arrest of CTLs. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Hypoxia and mitochondrial uncoupling interfered with CTL migration in vitro and, finally, highly vascular tumors grown in a matrix containing pro-angiogenic factors were more easily rejected after CTL injection, displayed delayed growth in untreated mice but were not affected in immunodeficient mice. Conclusions: We suggest that CTLs depend on oxygen for intratumoral locomotion and cytolysis. Incomplete vascularization promotes patchy infiltration and tumor escape; tumor immunotherapy may thus benefit from normal and extensive tumor angiogenesis Citation Format: Guy Shakhar, Tali Feferman, Yoav Manaster, Anat Hutzler. Live imaging reveals hypoxia to be a limiting factor in CTL-mediated tumor rejection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B84.
FDA-approved anti–PD-L1 monoclonal antibodies (mAbs) bear the IgG1 isotype, whose scaffolds are e... more FDA-approved anti–PD-L1 monoclonal antibodies (mAbs) bear the IgG1 isotype, whose scaffolds are either wild-type (e.g., avelumab) or Fc-mutated and lacking Fcγ receptor (FcγR) engagement (e.g., atezolizumab). It is unknown whether variation in the ability of the IgG1 Fc region to engage FcγRs renders mAbs with superior therapeutic activity. In this study, we used humanized FcγR mice to study the contribution of FcγR signaling to the antitumor activity of human anti–PD-L1 mAbs and to identify an optimal human IgG scaffold for PD-L1 mAbs. We observed similar antitumor efficacy and comparable tumor immune responses in mice treated with anti–PD-L1 mAbs with wild-type and Fc-mutated IgG scaffolds. However, in vivo antitumor activity of the wild-type anti–PD-L1 mAb avelumab was enhanced by combination treatment with an FcγRIIB-blocking antibody, which was co-administered to overcome the suppressor function of FcγRIIB in the tumor microenvironment (TME). We performed Fc glycoengineering to remove the fucose subunit from the Fc-attached glycan of avelumab to enhance its binding to the activating FcγRIIIA. Treatment with the Fc-afucosylated version of avelumab also enhanced antitumor activity and induced stronger antitumor immune responses compared with the parental IgG. The enhanced effect by afucosylated PD-L1 antibody was dependent on neutrophils and associated with decreased frequencies of PD-L1 + myeloid cells and increased infiltration of T cells in the TME. Our data reveal that the current design of FDA-approved anti–PD-L1 mAbs does not optimally harness FcγR pathways and suggest two strategies to enhance FcγR engagement to optimize anti–PD-L1 immunotherapy.
OBJECTIVE: To elucidate the requirement for T-regulatory cells (Tregs) in the therapeutic activit... more OBJECTIVE: To elucidate the requirement for T-regulatory cells (Tregs) in the therapeutic activity of glatiramer acetate (GA) in the animal model of multiple sclerosis - experimental autoimmune encephalomyelitis (EAE). BACKGROUND: GA, a therapeutic agent for MS, induces a broad immunomodulatory effect on various immune subsets, resulting in a shift towards anti-inflammatory responses. Several studies demonstrated an effect of GA on Tregs, however, the role of these cells in its therapeutic effect has not been clarified. DESIGN/METHODS: Tregs were identified immunohistochemically in the CNS of EAE-induced mice. The ability of GA to ameliorate EAE was tested in Foxp3GFPLuciDTR-4 transgenic mice, in which Tregs express diphtheria toxin (DT) receptor, facilitating their selective depletion. GA or PBS were injected for 7 days following EAE induction. DT was injected one and two days after treatment conclusion. Flow cytometry was used to verify Tregs depletion. RESULTS: Immunohistological analysis revealed 2-fold elevation in the number of CD3+Foxp3+ Tregs in the CNS of EAE-mice following GA treatment. GA drastically reduced disease manifestation in wild type mice, and its effect was not abrogated by DT injection (maximal clinical score 0.5 in GA-treated compared to 2.9 in untreated mice). In mice carrying the Foxp3GFPLuciDTR-4 transgene injected with DT, GA was less effective than in wild type mice. Yet, even in these mice, the clinical score of the GA-treated was lower than in the untreated mice (1.6 and 3.0, respectively). CONCLUSIONS: GA induces elevation of Tregs. Selective depletion of Tregs reduces but does not eliminate the ability of GA to ameliorate EAE. These findings support the role of Tregs, but not in an exclusive fashion, in the therapeutic effect of GA. Study Supported by: Teva Disclosure: Dr. Aharoni has received research support from Teva Neuroscience. Dr. Tali has nothing to disclose. Dr. Shakhar has nothing to disclose. Dr. Sela has received royalty payments from Teva Neuroscience. Dr. Arnon has received royalty payments from Teva.
Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated... more Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6–8 weeks post-transplant, with donor derived structures including epithelial, endothelial, and mesenchymal ...
The acetylcholine receptor (AChR) is the major autoantigen in the antibody-mediated disease myast... more The acetylcholine receptor (AChR) is the major autoantigen in the antibody-mediated disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). This study demonstrates that rats immunized with a recombinant fragment corresponding to the normally exposed extracellular region of the rat AChR alpha-subunit first develop antibodies to the injected extracellular portion only, but later develop antibodies to intracellular cytoplasmic epitopes of AChR. The presence of autoantibodies to intracellular epitopes seems to be correlated with development of clinical signs of disease. We propose that a similar process of epitope spreading may take place in the natural course of myasthenia.
We have previously demonstrated that the chemokine IFN-gamma inducible protein 10 (IP-10) and its... more We have previously demonstrated that the chemokine IFN-gamma inducible protein 10 (IP-10) and its receptor CXCR3, are overexpressed in myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG). We now studied the potential of modulating rat EAMG by interference in CXCR3/IP-10 signaling. Two different approaches were used: 1) blocking IP-10 by IP-10-specific antibodies and 2) inhibiting the CXCR3 chemokine receptor by a CXCR3 antagonist. Treatment by either of these reagents led to suppression of EAMG suggesting that inhibition of CXCR3/IP-10 signaling can be considered as a potential treatment modality for MG.
Protein databases were searched for microbial sequences that bear amino acid similarities with id... more Protein databases were searched for microbial sequences that bear amino acid similarities with identified T- or B-cell epitopes within the human alpha-subunit of acetylcholine receptor (AChR). One peptide, derived from Haemophilus influenzae, exhibits 50% homology to an identified T-cell epitope of AChR alpha-subunit. This peptide was shown to have a protective effect in experimental autoimmune myasthenia gravis (EAMG). Pretreatment of rats with the mimicry peptide attenuated the induction and progression of EAMG. These effects were accompanied by a reduced T-cell response to AChR, diminished IL-2, IL-12, IFN-gamma and IL-4 levels, as well as decreased humoral response to self-AChR.
Background: [corrected] Intravenous immunoglobulin administration has been beneficially used for ... more Background: [corrected] Intravenous immunoglobulin administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis, although its mode of action and active components have not yet been fully identified. Objectives: To isolate from IVIg a disease-specific fraction involved in the therapeutic activity in myasthenia and to identify its properties and function. Results: IVIg administration in experimental autoimmune MG results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of IVIg on columns of IgG from rats with EAMG or from MG patients resulted in depletion of the suppressive activity that IVIg has on rat EAMG. Moreover, the minute amounts of IgG fractions eluted from the EAMG or MG-specific columns retained the immunosuppressive activity of IVIg. Conclusions: Our study supports the notion that the therapeutic effect of IVIg is mediated by a minor disease-specific immunoglobulin fraction that is present in IVIg and is essential for its therapeutic activity.
Annals of the New York Academy of Sciences, Sep 1, 2007
Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of ... more Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis (MG). We have demonstrated that IVIG administration in experimental autoimmune MG (EAMG) results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of pooled human immunoglobulins (IVIGs) on immobilized IgG, isolated from rats with EAMG, results in a complete depletion of the suppressive activity of the IVIG. Moreover, the eluate from this EAMG-specific antibody column retains the immunosuppressive activity of IVIG. This study supports the notion that the therapeutic effect of IVIGs is mediated by an antigen-specific anti-immunoglobulin (anti-idiotypic) activity that is essential for its suppressive activity.
Annals of the New York Academy of Sciences, Jun 1, 2008
Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of ... more Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis (MG). We have demonstrated that IVIG administration in experimental autoimmune MG (EAMG) results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of pooled human immunoglobulins (IVIG) on immobilized IgG, isolated from rats with EAMG or from MG patients, results in a depletion of the suppressive activity of the IVIG. Moreover, reconstitution of the activity‐depleted IVIG with the eluted minute IVIG fractions that had been adsorbed onto the EAMG‐ or MG‐specific columns recovers the depleted immunosuppressive activity. This study supports the notion that the therapeutic effect of IVIG is mediated by an antigen‐specific anti‐immunoglobulin (anti‐idiotypic) activity that is essential for its suppressive activity.
Intravenous immunoglobulin (IVIG) treatment is beneficially used in autoimmune disorders includin... more Intravenous immunoglobulin (IVIG) treatment is beneficially used in autoimmune disorders including myasthenia gravis (MG) although its mode of action and active components are still not fully identified. In an attempt to isolate from IVIG a disease-specific suppressive fraction, IVIG was passed on columns of IgG from rats with experimental autoimmune MG (EAMG) or from MG patients. These chromatographies resulted in depletion of the suppressive activity of IVIG on rat EAMG whereas the minute amounts of IgG fractions eluted from the EAMG- or MG-specific columns retained the immunosuppressive activity of IVIG. These results demonstrate that a minor disease-specific immunoglobulin fraction present in IVIG is essential for its suppressive activity.
Introduction: Immunotherapies that harness CTLs to eradicate solid tumors, melanoma in particular... more Introduction: Immunotherapies that harness CTLs to eradicate solid tumors, melanoma in particular, have re-emerged as potent clinical options in recent years. Improving the dispersion and cytotoxicity of intratumoral CTLs may further benefit patients. To gain insight into the regulatory processes that limit CTL activity in melanoma we investigated the influence of PD-1 signaling and Tregs on CTL behavior. Methodology: To mimic adoptive cell therapy we transferred mice bearing B16.OVA melanoma tumors with OT-I CTLs. To study Treg-mediated regulation, we used FOXP3-GFP-LuciDTR-4 host mice, in which 95% of the cells expressing Foxp3 could be depleted using diphtheria toxin. To study PD-1 mediated regulation we injected anti PD-1 to WT host mice. Tumor rejection was monitored for 2-5 weeks. Two-photon imaging of live mice was used to follow the behavior of CTLs and Tregs within tumors. Time-lapse confocal microscopy was used to follow the influence of PD-1 signaling on the behavior of CTLs and their interaction with B16-Ova cells. Results: Tumor rejection was closely regulated by Tregs as tumors were densely infiltrated by these cells and their depletion dramatically accelerated tumor rejection. Interactions between CTLs and Tregs seemed indirect, as they did not form conjugates. Following their transfer, antigen-specific OT-I CTLs, as well as polyclonal CTLs, entered murine tumors through selected ICAM-decorated intratumoral vessels. As CTLs accumulated, Treg numbers in the tumors dropped and CTLs could be observed conjugating tumor cells and killing them. Treatment with anti PD-1 increased CTL velocities and accelerated killing of B16.OVA cells in vitro, and was mirrored in vivo by enhanced tumor rejection. Whereas CTL migration and cytolysis was limited to oxygenated tumor areas within 50 µm of flowing blood vessels, Tregs exhibited higher velocities, were not affected by the proximity of blood vessels, and penetrated deeper into avascular areas. Conclusions: Our findings suggest that regulatory mechanisms influence CTL efficiency partly by affecting their migration. CTLs, more than Tregs, depend on oxygen for intratumoral locomotion and cytolysis. This disparity allows tumor cells to escape CTL lysis in deep hypoxic tumor niches where high Treg to CTL ratios favor their survival. Thus, differential motility and dependence on oxygen among CTLs and Tregs can dictate the efficiency of CTL-based therapies of melanoma. Citation Format: Tali Feferman, Natalia G. Zabolinsky, Yoav Manaster, Anat Hutzler, Guy Shakhar. Imaging regulatory mechanisms that limit intratumoral CTL function in a mouse melanoma model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B06.
Background: One of the obstacles to successful immunotherapy by CTLs is thought to be poor vascul... more Background: One of the obstacles to successful immunotherapy by CTLs is thought to be poor vascularization of tumors that leads to sub-optimal tumor infiltration. In contrast, the effects of disrupted vascularization and the hypoxia it introduces on CTL migration and function within tumors have not been investigated. Methods: To study whether tumor vascularity can shape anti-tumor CTL responses, we combined immunohistochemistry of human melanoma samples with dynamic two-photon imaging within B16-OVA tumors in live mice. Results: In patients, CD8 T cells were twofold denser around peripheral vessels than around deep vessels, and fourfold denser than in avascular areas. This pattern was clearest in poorly-infiltrated tumors. Correspondingly, in mice, OT-I CTLs preferentially infiltrated B16-OVA tumors through peripheral ICAM1+ vessels. CTLs remained initially at their entry areas, crawling vigorously only within 50 µm of flowing blood vessels. Gradually, CTLs formed dense perivascular swarms, producing locally-high CTL-to-target ratios at their advancing front. These swarms advanced through the tumors, leading to patchy elimination of tumor cells. Target killing was antigen-specific, contact-dependent and perforin-mediated, and required several CTLs to engage one target cells to be effective. Several findings suggest that CTL confinement to perivascular areas is oxygen-dependent: occlusion of intratumoral blood vessels resulted in immediate arrest of CTLs. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Hypoxia and mitochondrial uncoupling interfered with CTL migration in vitro and, finally, highly vascular tumors grown in a matrix containing pro-angiogenic factors were more easily rejected after CTL injection, displayed delayed growth in untreated mice but were not affected in immunodeficient mice. Conclusions: We suggest that CTLs depend on oxygen for intratumoral locomotion and cytolysis. Incomplete vascularization promotes patchy infiltration and tumor escape; tumor immunotherapy may thus benefit from normal and extensive tumor angiogenesis Citation Format: Guy Shakhar, Tali Feferman, Yoav Manaster, Anat Hutzler. Live imaging reveals hypoxia to be a limiting factor in CTL-mediated tumor rejection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B84.
FDA-approved anti–PD-L1 monoclonal antibodies (mAbs) bear the IgG1 isotype, whose scaffolds are e... more FDA-approved anti–PD-L1 monoclonal antibodies (mAbs) bear the IgG1 isotype, whose scaffolds are either wild-type (e.g., avelumab) or Fc-mutated and lacking Fcγ receptor (FcγR) engagement (e.g., atezolizumab). It is unknown whether variation in the ability of the IgG1 Fc region to engage FcγRs renders mAbs with superior therapeutic activity. In this study, we used humanized FcγR mice to study the contribution of FcγR signaling to the antitumor activity of human anti–PD-L1 mAbs and to identify an optimal human IgG scaffold for PD-L1 mAbs. We observed similar antitumor efficacy and comparable tumor immune responses in mice treated with anti–PD-L1 mAbs with wild-type and Fc-mutated IgG scaffolds. However, in vivo antitumor activity of the wild-type anti–PD-L1 mAb avelumab was enhanced by combination treatment with an FcγRIIB-blocking antibody, which was co-administered to overcome the suppressor function of FcγRIIB in the tumor microenvironment (TME). We performed Fc glycoengineering to remove the fucose subunit from the Fc-attached glycan of avelumab to enhance its binding to the activating FcγRIIIA. Treatment with the Fc-afucosylated version of avelumab also enhanced antitumor activity and induced stronger antitumor immune responses compared with the parental IgG. The enhanced effect by afucosylated PD-L1 antibody was dependent on neutrophils and associated with decreased frequencies of PD-L1 + myeloid cells and increased infiltration of T cells in the TME. Our data reveal that the current design of FDA-approved anti–PD-L1 mAbs does not optimally harness FcγR pathways and suggest two strategies to enhance FcγR engagement to optimize anti–PD-L1 immunotherapy.
OBJECTIVE: To elucidate the requirement for T-regulatory cells (Tregs) in the therapeutic activit... more OBJECTIVE: To elucidate the requirement for T-regulatory cells (Tregs) in the therapeutic activity of glatiramer acetate (GA) in the animal model of multiple sclerosis - experimental autoimmune encephalomyelitis (EAE). BACKGROUND: GA, a therapeutic agent for MS, induces a broad immunomodulatory effect on various immune subsets, resulting in a shift towards anti-inflammatory responses. Several studies demonstrated an effect of GA on Tregs, however, the role of these cells in its therapeutic effect has not been clarified. DESIGN/METHODS: Tregs were identified immunohistochemically in the CNS of EAE-induced mice. The ability of GA to ameliorate EAE was tested in Foxp3GFPLuciDTR-4 transgenic mice, in which Tregs express diphtheria toxin (DT) receptor, facilitating their selective depletion. GA or PBS were injected for 7 days following EAE induction. DT was injected one and two days after treatment conclusion. Flow cytometry was used to verify Tregs depletion. RESULTS: Immunohistological analysis revealed 2-fold elevation in the number of CD3+Foxp3+ Tregs in the CNS of EAE-mice following GA treatment. GA drastically reduced disease manifestation in wild type mice, and its effect was not abrogated by DT injection (maximal clinical score 0.5 in GA-treated compared to 2.9 in untreated mice). In mice carrying the Foxp3GFPLuciDTR-4 transgene injected with DT, GA was less effective than in wild type mice. Yet, even in these mice, the clinical score of the GA-treated was lower than in the untreated mice (1.6 and 3.0, respectively). CONCLUSIONS: GA induces elevation of Tregs. Selective depletion of Tregs reduces but does not eliminate the ability of GA to ameliorate EAE. These findings support the role of Tregs, but not in an exclusive fashion, in the therapeutic effect of GA. Study Supported by: Teva Disclosure: Dr. Aharoni has received research support from Teva Neuroscience. Dr. Tali has nothing to disclose. Dr. Shakhar has nothing to disclose. Dr. Sela has received royalty payments from Teva Neuroscience. Dr. Arnon has received royalty payments from Teva.
Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated... more Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6–8 weeks post-transplant, with donor derived structures including epithelial, endothelial, and mesenchymal ...
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