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Mouse Invariant Monoclonal Antibody NKT14: A Novel Tool to Manipulate iNKT Cell Function In Vivo.

Scheuplein F, Lamont DJ, Poynter ME, Boyson JE, Serreze D, Lundblad LK, Mashal R, Schaub R - PLoS ONE (2015)

Bottom Line: Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery.This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth.These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.

View Article: PubMed Central - PubMed

Affiliation: NKT Therapeutics, Inc., Waltham, MA, United States of America.

ABSTRACT
Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.

No MeSH data available.


Related in: MedlinePlus

C57BL/6 splenocytes were stained with NKT14 or isotype control followed by a secondary anti mouse IgG2a monoclonal and αGalCer loaded CD1d Tetramer (A) The panel on the far right demonstrates co-binding of NKT14 and CD1d Tetramer. C57BL/6 mice (n = 3 per group) were injected i.p. with 50 μg NKT14 or isotype control. Mice were euthanized 24 hours after dosing and splenocytes were prepared, counted and stained for CD3, CD4, CD8 and aGalCer-loaded CD1d Tetramer to assess specificity of iNKT cell depletion. Data are representative of > 10 experiments (B). The CD4/CD8 ratio panel was gated on CD3+ cells. C57BL/6 (n = 3 per group) mice were injected with 2μg aGalCer, 50 μg NKT14m or isotype control. 2 hours post dosing mice were euthanized, splenocytes were prepared and stained for CD3 and αGalCer loaded CD1d tetramers to identify iNKT cells, washed, fixed, permeabilized and stained for intracellular IFN-Gamma. Representative FACS plots are shown as well as intracellular IFN-γ measurement across treatment group (C). Data are representative of >5 experiments.
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pone.0140729.g001: C57BL/6 splenocytes were stained with NKT14 or isotype control followed by a secondary anti mouse IgG2a monoclonal and αGalCer loaded CD1d Tetramer (A) The panel on the far right demonstrates co-binding of NKT14 and CD1d Tetramer. C57BL/6 mice (n = 3 per group) were injected i.p. with 50 μg NKT14 or isotype control. Mice were euthanized 24 hours after dosing and splenocytes were prepared, counted and stained for CD3, CD4, CD8 and aGalCer-loaded CD1d Tetramer to assess specificity of iNKT cell depletion. Data are representative of > 10 experiments (B). The CD4/CD8 ratio panel was gated on CD3+ cells. C57BL/6 (n = 3 per group) mice were injected with 2μg aGalCer, 50 μg NKT14m or isotype control. 2 hours post dosing mice were euthanized, splenocytes were prepared and stained for CD3 and αGalCer loaded CD1d tetramers to identify iNKT cells, washed, fixed, permeabilized and stained for intracellular IFN-Gamma. Representative FACS plots are shown as well as intracellular IFN-γ measurement across treatment group (C). Data are representative of >5 experiments.

Mentions: Leveraging EUREKA Therapeutics’ proprietary human phage display library we identified a mouse invariant TCR specific antibody clone. Full length antibodies were made by cloning them in frame with either murine wild type IgG2a Fc to obtain a depleting antibody (NKT14) or an IgG2a with 4 point mutations to the Fc portion (L235E, E318A, K320A and K322A) to greatly reduce antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity CDC function [18] for iNKT cell activation (NKT14m). NKT14 specifically binds to mouse iNKT cells identified by binding of αGalCer loaded CD1d Tetramers. Fig 1A shows co-binding of NKT14 to CD1d Tetramer positive iNKT cells in C57BL/6 splenocytes. There is no competition between CD1d Tetramer and NKT14, indicating different binding epitopes. We have tested multiple inbred mouse strains and confirmed this high specificity in the BALB/c, NOD, DBA, C3H, NZW, NZWxNZB, AKR and SJL strains (S1 Fig). NKT14 specifically depletes iNKT cells in vivo for approximately 3 weeks. 50 μg of NKT14 injected i.p. depleted iNKT cells without altering splenic lymphocyte count, T cell numbers or CD4/CD8 ratio (Fig 1B). Conversely, (and analogous to the αGalCer agonist) 50 μg of NKT14m injected i.v. rapidly activated iNKT cells, as indicated by measurable intracellular IFN-Gamma (Fig 1C), further upregulation of CD69 and upregulation of CD25 (S2 Fig).


Mouse Invariant Monoclonal Antibody NKT14: A Novel Tool to Manipulate iNKT Cell Function In Vivo.

Scheuplein F, Lamont DJ, Poynter ME, Boyson JE, Serreze D, Lundblad LK, Mashal R, Schaub R - PLoS ONE (2015)

C57BL/6 splenocytes were stained with NKT14 or isotype control followed by a secondary anti mouse IgG2a monoclonal and αGalCer loaded CD1d Tetramer (A) The panel on the far right demonstrates co-binding of NKT14 and CD1d Tetramer. C57BL/6 mice (n = 3 per group) were injected i.p. with 50 μg NKT14 or isotype control. Mice were euthanized 24 hours after dosing and splenocytes were prepared, counted and stained for CD3, CD4, CD8 and aGalCer-loaded CD1d Tetramer to assess specificity of iNKT cell depletion. Data are representative of > 10 experiments (B). The CD4/CD8 ratio panel was gated on CD3+ cells. C57BL/6 (n = 3 per group) mice were injected with 2μg aGalCer, 50 μg NKT14m or isotype control. 2 hours post dosing mice were euthanized, splenocytes were prepared and stained for CD3 and αGalCer loaded CD1d tetramers to identify iNKT cells, washed, fixed, permeabilized and stained for intracellular IFN-Gamma. Representative FACS plots are shown as well as intracellular IFN-γ measurement across treatment group (C). Data are representative of >5 experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608835&req=5

pone.0140729.g001: C57BL/6 splenocytes were stained with NKT14 or isotype control followed by a secondary anti mouse IgG2a monoclonal and αGalCer loaded CD1d Tetramer (A) The panel on the far right demonstrates co-binding of NKT14 and CD1d Tetramer. C57BL/6 mice (n = 3 per group) were injected i.p. with 50 μg NKT14 or isotype control. Mice were euthanized 24 hours after dosing and splenocytes were prepared, counted and stained for CD3, CD4, CD8 and aGalCer-loaded CD1d Tetramer to assess specificity of iNKT cell depletion. Data are representative of > 10 experiments (B). The CD4/CD8 ratio panel was gated on CD3+ cells. C57BL/6 (n = 3 per group) mice were injected with 2μg aGalCer, 50 μg NKT14m or isotype control. 2 hours post dosing mice were euthanized, splenocytes were prepared and stained for CD3 and αGalCer loaded CD1d tetramers to identify iNKT cells, washed, fixed, permeabilized and stained for intracellular IFN-Gamma. Representative FACS plots are shown as well as intracellular IFN-γ measurement across treatment group (C). Data are representative of >5 experiments.
Mentions: Leveraging EUREKA Therapeutics’ proprietary human phage display library we identified a mouse invariant TCR specific antibody clone. Full length antibodies were made by cloning them in frame with either murine wild type IgG2a Fc to obtain a depleting antibody (NKT14) or an IgG2a with 4 point mutations to the Fc portion (L235E, E318A, K320A and K322A) to greatly reduce antibody dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity CDC function [18] for iNKT cell activation (NKT14m). NKT14 specifically binds to mouse iNKT cells identified by binding of αGalCer loaded CD1d Tetramers. Fig 1A shows co-binding of NKT14 to CD1d Tetramer positive iNKT cells in C57BL/6 splenocytes. There is no competition between CD1d Tetramer and NKT14, indicating different binding epitopes. We have tested multiple inbred mouse strains and confirmed this high specificity in the BALB/c, NOD, DBA, C3H, NZW, NZWxNZB, AKR and SJL strains (S1 Fig). NKT14 specifically depletes iNKT cells in vivo for approximately 3 weeks. 50 μg of NKT14 injected i.p. depleted iNKT cells without altering splenic lymphocyte count, T cell numbers or CD4/CD8 ratio (Fig 1B). Conversely, (and analogous to the αGalCer agonist) 50 μg of NKT14m injected i.v. rapidly activated iNKT cells, as indicated by measurable intracellular IFN-Gamma (Fig 1C), further upregulation of CD69 and upregulation of CD25 (S2 Fig).

Bottom Line: Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery.This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth.These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.

View Article: PubMed Central - PubMed

Affiliation: NKT Therapeutics, Inc., Waltham, MA, United States of America.

ABSTRACT
Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation.

No MeSH data available.


Related in: MedlinePlus