Limits...
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

A) BALB/c female mice (8 per group) were treated with NKT14 (NKT-14/OVA) three days before being sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23. AHR was determined by methacholine challenge two days later. OVA/OVA are non-treated positive control mice and PBS/OVA mice were sham-sensitized negative controls. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly reduced the response in Rn compared with OVA/OVA (p<0.001); NKT-14 treatment also reduced the response in G (p<0.0001) and H, p<0.01) compared with positive control (OVA/OVA). B) Mice were sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23 and then again once after a 30 day recovery phase.AHR was determined by methacholine challenge two days later. iNKT cells were depleted with NKT14 three days before the final antigen challenge. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly increased the response in Rn compared with sham control (PBS/OVA) (p<0.01), whereas positive control (OVA/OVA) was significantly increased over PBS/OVA (p<0.05). However, the response in G was significantly reduced by NKT-14 compared with OVA/OVA (p<0.001). The response in H was also reduced by NKT-14 compared with OVA/OVA (p<0.001) Positive control group (OVA/OVA) was significantly increased over the sham control group (PBS/OVA) in G and H, p<0.0001 and p<0.001 respectively. Depletion of lung iNKT cells was confirmed by flow cytometry after euthanasia, but no differences in CD4+ or CD8+ cells were noted (S3 Fig). Analysis of Broncho alveolar fluid (BALF) found no differences in total cells across treatment groups. BALF eosinophils increased as both a percent and as absolute counts following sensitization. A small, but significant elevation of eosinophils was observed in NKT-14 treated mice compared to OVA/OVA mice (Data not shown).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608835&req=5

pone.0140729.g004: A) BALB/c female mice (8 per group) were treated with NKT14 (NKT-14/OVA) three days before being sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23. AHR was determined by methacholine challenge two days later. OVA/OVA are non-treated positive control mice and PBS/OVA mice were sham-sensitized negative controls. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly reduced the response in Rn compared with OVA/OVA (p<0.001); NKT-14 treatment also reduced the response in G (p<0.0001) and H, p<0.01) compared with positive control (OVA/OVA). B) Mice were sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23 and then again once after a 30 day recovery phase.AHR was determined by methacholine challenge two days later. iNKT cells were depleted with NKT14 three days before the final antigen challenge. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly increased the response in Rn compared with sham control (PBS/OVA) (p<0.01), whereas positive control (OVA/OVA) was significantly increased over PBS/OVA (p<0.05). However, the response in G was significantly reduced by NKT-14 compared with OVA/OVA (p<0.001). The response in H was also reduced by NKT-14 compared with OVA/OVA (p<0.001) Positive control group (OVA/OVA) was significantly increased over the sham control group (PBS/OVA) in G and H, p<0.0001 and p<0.001 respectively. Depletion of lung iNKT cells was confirmed by flow cytometry after euthanasia, but no differences in CD4+ or CD8+ cells were noted (S3 Fig). Analysis of Broncho alveolar fluid (BALF) found no differences in total cells across treatment groups. BALF eosinophils increased as both a percent and as absolute counts following sensitization. A small, but significant elevation of eosinophils was observed in NKT-14 treated mice compared to OVA/OVA mice (Data not shown).

Mentions: To study the role of iNKT cells in a model of allergic airway inflammation and airway hyperreactivity (AHR) we treated BALB/c mice (n = 8/ group) with the iNKT cell depleting antibody NKT14 either before the sensitization phase, or just days before antigen re-challenge. When mice were treated before the sensitization phase, AHR to inhaled methacholine challenge was completely abrogated (Fig 4A). Conversely, untreated, but sensitized animals showed a significant AHR reaction. However, when iNKT cell depletion occurred in sensitized mice, briefly before the final antigen challenge, iNKT cell depletion completely failed to protect the mice from AHR in the conducting airways but significantly reduced AHR in the periphery of the airways as evidenced by reductions in G and H (Fig 4B).


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)

A) BALB/c female mice (8 per group) were treated with NKT14 (NKT-14/OVA) three days before being sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23. AHR was determined by methacholine challenge two days later. OVA/OVA are non-treated positive control mice and PBS/OVA mice were sham-sensitized negative controls. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly reduced the response in Rn compared with OVA/OVA (p<0.001); NKT-14 treatment also reduced the response in G (p<0.0001) and H, p<0.01) compared with positive control (OVA/OVA). B) Mice were sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23 and then again once after a 30 day recovery phase.AHR was determined by methacholine challenge two days later. iNKT cells were depleted with NKT14 three days before the final antigen challenge. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly increased the response in Rn compared with sham control (PBS/OVA) (p<0.01), whereas positive control (OVA/OVA) was significantly increased over PBS/OVA (p<0.05). However, the response in G was significantly reduced by NKT-14 compared with OVA/OVA (p<0.001). The response in H was also reduced by NKT-14 compared with OVA/OVA (p<0.001) Positive control group (OVA/OVA) was significantly increased over the sham control group (PBS/OVA) in G and H, p<0.0001 and p<0.001 respectively. Depletion of lung iNKT cells was confirmed by flow cytometry after euthanasia, but no differences in CD4+ or CD8+ cells were noted (S3 Fig). Analysis of Broncho alveolar fluid (BALF) found no differences in total cells across treatment groups. BALF eosinophils increased as both a percent and as absolute counts following sensitization. A small, but significant elevation of eosinophils was observed in NKT-14 treated mice compared to OVA/OVA mice (Data not shown).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608835&req=5

pone.0140729.g004: A) BALB/c female mice (8 per group) were treated with NKT14 (NKT-14/OVA) three days before being sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23. AHR was determined by methacholine challenge two days later. OVA/OVA are non-treated positive control mice and PBS/OVA mice were sham-sensitized negative controls. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly reduced the response in Rn compared with OVA/OVA (p<0.001); NKT-14 treatment also reduced the response in G (p<0.0001) and H, p<0.01) compared with positive control (OVA/OVA). B) Mice were sensitized on days 0 and 14 with i.p. injections of OVA in ImjectAlum and then exposed to inhalational challenges with 1% OVA or control saline on days 21, 22, and 23 and then again once after a 30 day recovery phase.AHR was determined by methacholine challenge two days later. iNKT cells were depleted with NKT14 three days before the final antigen challenge. At the methacholine dose of 25 mg/ml NKT-14 treatment (NKT-14/OVA) significantly increased the response in Rn compared with sham control (PBS/OVA) (p<0.01), whereas positive control (OVA/OVA) was significantly increased over PBS/OVA (p<0.05). However, the response in G was significantly reduced by NKT-14 compared with OVA/OVA (p<0.001). The response in H was also reduced by NKT-14 compared with OVA/OVA (p<0.001) Positive control group (OVA/OVA) was significantly increased over the sham control group (PBS/OVA) in G and H, p<0.0001 and p<0.001 respectively. Depletion of lung iNKT cells was confirmed by flow cytometry after euthanasia, but no differences in CD4+ or CD8+ cells were noted (S3 Fig). Analysis of Broncho alveolar fluid (BALF) found no differences in total cells across treatment groups. BALF eosinophils increased as both a percent and as absolute counts following sensitization. A small, but significant elevation of eosinophils was observed in NKT-14 treated mice compared to OVA/OVA mice (Data not shown).
Mentions: To study the role of iNKT cells in a model of allergic airway inflammation and airway hyperreactivity (AHR) we treated BALB/c mice (n = 8/ group) with the iNKT cell depleting antibody NKT14 either before the sensitization phase, or just days before antigen re-challenge. When mice were treated before the sensitization phase, AHR to inhaled methacholine challenge was completely abrogated (Fig 4A). Conversely, untreated, but sensitized animals showed a significant AHR reaction. However, when iNKT cell depletion occurred in sensitized mice, briefly before the final antigen challenge, iNKT cell depletion completely failed to protect the mice from AHR in the conducting airways but significantly reduced AHR in the periphery of the airways as evidenced by reductions in G and H (Fig 4B).

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